JP2023090751A - CD14 antagonist antibodies for treating neurodegenerative diseases - Google Patents

Abstract

To provide agents and methods for treating onset or progression of neurodegenerative diseases.SOLUTION: The invention relates to a CD14 antagonist for use in the treatment of the onset or progress of a neurodegenerative disease including motor neuron disease (MND) and dementia or related symptoms. The invention further provides a composition comprising such an agent.SELECTED DRAWING: None

Description

This application is subject to Australian Provisional Application No. 2017901462 entitled "Agents for treating or preventing motor neurone disease and uses therefor" filed on 21 April 2017 and "Agents for Australian Provisional Application No. 2018900762 entitled "Treatment Disease and Uses Therefor", the contents of which are incorporated herein by reference in their entirety.

The present invention relates generally to agents and methods for treating the development or progression of neurodegenerative diseases. In particular, the invention relates to CD14 antagonists for use in treating the development or progression of neurodegenerative diseases such as motor neuron disease (MND) and dementia diseases or related conditions. The invention further provides compositions comprising such agents.

Neurodegenerative diseases are highly debilitating conditions characterized by progressive loss of neuronal structure and function, ultimately leading to neuronal death and having a detrimental impact on quality of life and ultimately lead to premature death. It also has a great social impact on surrounding family members and communities. As the population ages, the palliative care needed for patients with neurodegenerative disorders is becoming a significant health cost to the community. In the United States alone, the management and lost productivity of patients with neurodegenerative diseases is tens of billions of dollars annually. The ALS Therapy Development Institute of Cambridge, Massachusetts estimates the number of motor neuron disease (MND) patients to be greater than 30,000 in the United States and at least 450,000 worldwide. The global cost of dementia in 2010 was estimated at $604 billion, which is approximately 1% of the world gross domestic product, and in the United States the Alzheimer's Association has reduced the cost of care for people with Alzheimer's disease (AD). estimated at $200 billion annually. If current trends continue, this cost is projected to grow to $1.1 trillion annually by 2050. Although extensive research is underway to find cures for these debilitating conditions, it may be decades before a successful treatment is found to reverse or prevent the effects of neurodegeneration. be.

The most common neurodegenerative diseases include MNDs such as amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP) ) and pseudobulbar palsy, and dementia (e.g., Alzheimer's disease, dementia with Lewy bodies, i.e. dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD), vascular dementia and frontotemporal type dementia (FTD)), Parkinson's disease and Huntington's disease.

Motor neuron disease (MND) is a devastating progressive disease that typically leads to death within 4-6 years of onset. The incidence of MND is fairly uniform at 2-3 cases per 100,000 persons per year, with a peak age of onset of 58-63 years for sporadic disease and 47-52 years for familial disease (Kiernan M.; C., et al.Lancet 377:942-955 (2011)). Pathologically, MND is characterized by loss of motor neurons in the motor cortex, brainstem and spinal cord. Patients typically present with progressive muscle wasting, paralysis, spasticity, and hyperreflexia, which ultimately lead to death through respiratory muscle failure (Zhao W., et al. Glia 58:231-243). (2010)). Treatment options for MND are extremely limited. A number of therapies have demonstrated efficacy in rodent models and appeared to be effective in small Phase 2 clinical trials, but in the last 23 years riluzole (Rilutek®/Teglutik®) ) and edaravone (Radicava®/Radicut®) have received US Food and Drug Administration (FDA) approval. Both have limited efficacy and extend patient survival by 3-4 months without improving quality of life. Another drug currently in Phase III trials is Genervon's drug candidate GM604, an endogenous embryonic tyrosine kinase motor neuron trophic factor that binds to insulin, IGF1 and IGF2 receptors in the human nervous system. (mononeuronotrophic factor) modulator. Although it has shown some positive effects on amyotrophic lateral sclerosis (ALS) symptoms (the most common form of MND) in clinical trials, the drug is, in the company's own words, a "cure" on its own. do not give Consistent failures of curative ALS trials and a 99.6% failure rate of AD trials indicate that single/gene-targeted reductionism is the widespread and dominant drug development paradigm for ALS and other neurological and neurodegenerative multifactorial Evidence that sexually transmitted diseases cannot be cured. This means that there is considerable room for improvement in therapeutic development of treatments for neurodegenerative diseases and their symptoms, highlighting an unmet medical need.

ALS accounts for about 60-70% of all MND cases. In the United States, ALS is commonly known as "Lou Gehrig's disease" and affects about 7 out of 100,000 people. There are two forms of ALS, familial and sporadic, in which subjects present with inherited mutations in genes. Genes with mutations associated with ALS (and other forms of MND) include C9ORF72, the most common genetic cause of ALS, Cu/Zn superoxide dismutase (SOD1), NEK1, TAR DNA binding protein 43 (TDP-43), Fused in sarcoma (FUS) and ubiquilin-2 (UBQLN2). VCP (valosin-containing protein), ALS2 (arsine), SETX (senataxin), ANG (angiogenin), PFN1 (profilin-1), MATR3 (matrin-3), CHCHD10 (coiled-coil-helix-coiled-coil-helix domain containing 10 ), TUBA4A (tubulin, alpha 4A), TBK1 (TANK binding kinase 1), C21orf2 and OPTN (optineurin) have also been found in a small percentage of patients.

The causative nature of ALS is diverse, and in this respect the disease can be considered a spectrum disorder. ALS is characterized by rapidly progressive muscle wasting, dysarthria and dyspnea. Disconnection of muscles and neurons leads to respiratory failure after about 40 months. There is degeneration of upper and lower motor neurons leading to muscle weakness and atrophy due to the inability of muscles to function. Finally, complete weakness. Cognitive function is generally preserved, but muscle atrophy is sufficient to significantly reduce the patient's quality of life.

In recent years there has been a growing recognition of the importance of inflammation in the progression of neurological disease. The role of inflammation in neurological disease has long been recognized in multiple sclerosis, resulting in the introduction of immunomodulatory drugs with significant therapeutic impact (Ransohoff, R. M., et al. Nat. Rev. Neurol. 11, 134-142 (2015)).

Immune responses in the brain and spinal cord are primarily mediated by the brain's endogenous immune cells, the microglia. They serve as a first line of defense by responding to activation by pathological changes in the central nervous system (CNS), for example by releasing danger signals such as damage-associated molecular patterns (DAMPs) from injured tissue. It plays a key role in initiating the production of pro-inflammatory mediators and sustaining neuroinflammatory responses. Studies have shown microglial activation at sites of motor neuron injury in both MND patients and murine MND models, and there is widespread microglial activation detectable by PET scanning in the brains of live MND patients. (Turner M.R., et al. Neurobiol. Dis. 15:601-9 (2004); Corcia P., et al. PLoS One 7:6-12 (2012)). A correlation between the severity of upper motor neuron signs and the level of microglial activation in the motor cortex in patients with MND has also been demonstrated (Turner MR, et al. Neurobiol. Dis. 15:601). -9 (2004)). Mouse MND models also support a role for microglia in disease progression, with MND-activated microglia increasing with disease progression prior to disease onset and throughout end-stage disease (Henkel JS et al. Mol. Cell Neurosci 31:427-37 (2006); It points to supporting the active role of cells.

A number of DAMPs have been identified in the brains of Alzheimer's patients. These include HMGB1, S100 family members S100A9 and S100B, chromogranin, circulating DNA and heat shock proteins (HSPs). With the exception of HSPs, all of these DAMPs have been shown to be elevated in disease, physically associated with Aβ plaques and involved in ongoing neuroinflammation (Venegas, C. & Heneka, M.; T. J. Leukoc. Biol. 101, 87-98 (2017)). Stimulation of microglia by Aβ results in the initiation of numerous pro-inflammatory responses such as cytokine and chemokine production.

Microglial activation has also been associated with neurodegeneration in frontotemporal dementia (FTD) and Lewy body dementia (LBD), which comprises two closely related conditions, which are , dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD), the second most common cause of dementia in the elderly. These degenerative brain diseases are associated with abnormal clusters of a protein called alpha-synuclein. FTD is a group of related conditions that result from progressive degeneration of the temporal and frontal lobes of the brain and are characterized by deposition of TDP-43. These compartments of the brain play critical roles in decision-making, behavioral control, emotion and language. Both α-synuclein and TDP-43 can act as DAMPs. Microglial activation has also been associated with dopaminergic loss in early Parkinson's disease. This microglia activation is also inversely correlated with cognitive function, suggesting that it may be involved in disease pathology.

Diagnosis and therapy of diseases of the central nervous system may benefit from the application of highly specific monoclonal antibodies as drugs. However, one of the difficulties in developing treatments for neurodegenerative disorders is the problem of delivering drugs across the blood-brain barrier (BBB) to target sites in the CNS. This limits the effectiveness of drugs targeting brain diseases and makes it difficult to use biologic agents for brain diseases. Since the size of even small antibody fragments (Fv, Fab) severely limits attempts to increase delivery by passive diffusion across the intact BBB, the methods being evaluated are directed to cerebrospinal fluid or brain tissue. Attempts to circumvent the barrier by administration of a steroid, or to disrupt the barrier temporarily with a hypertonic solution. Both approaches require invasive techniques.

Given the severity of neurodegenerative diseases and the limited treatment options available, there is a need for agents and methods for treating neurodegenerative diseases.

The present invention provides, in part, that systemic administration of a CD14 antagonist antibody outside the central nervous system (CNS) of a subject with ALS will induce the production of proinflammatory mediators (e.g., cytokines) by cells in the periphery of the subject. inhibit and result in attenuation of disease progression and/or amelioration of symptoms of ALS in the CNS, thus addressing the causative signals involved in generating and sustaining the inflammatory process rather than treating secondary inflammatory effectors It arose from the decision that

CD14 is present in a soluble form (sCD14) and on various cells such as endothelial and epithelial cells, as well as immune cells such as macrophages, monocytes, Kupffer cells, neutrophils, dendritic cells and B cells. is a glycoprotein that exists both in a cell membrane-bound form (mCD14) on the surface of the cell (Jersmann, HPA, 2005. Immunol Cell Biol. 83: 462-467).

CD14 is a key molecule involved in mediating cell activation through the pattern recognition receptor (PRR) family, either derived from pathogens (pathogen-associated molecular patterns (PAMPs)) or via Toll-like receptors ( involved in the recognition of danger signals as a consequence of intrinsic tissue damage or stress (damage-associated molecular patterns (DAMPs)) by TLRs) and subsequent initiation of inflammatory cascades. CD14 is an exemplary PAMP, also known as a receptor for lipopolysaccharide (LPS), also known as Gram-negative bacterial endotoxin, which accepts LPS from LPS-binding protein (LBP) in the blood to form a complex. Form. LPS also binds to sCD14 and the resulting complex can induce mCD14-independent production of pro-inflammatory mediators such as pro-inflammatory cytokines. Culture of human CD4+CD45RO+ memory T cells with soluble CD14 has also been shown to be sufficient for upregulation of the retinoic acid-related orphan receptor gamma thymus and production of IL-17 (Ilarregui JM. , et al., Immunol.Cell Biol.94: (2016)).

In addition to LPS, a component of Gram-negative bacteria, CD14 is also a co-receptor for other PAMPs such as peptidoglycan and lipoteichoic acid in Gram-positive bacteria, lipoarabinomannan in mycobacteria and viral envelope proteins. works. CD14 also recognizes host-derived DAMP ligands, endogenous molecules released from stressed, damaged or dying cells as a result of tissue injury and inflammation associated with a wide range of inflammatory situations. DAMPs exert their activity through interactions with members of the TLR family, and CD14 is associated with SOD1, TDP-43, members of the heat shock protein family, as well as HMGB1, S100A and various misfoldings associated with neurodegenerative disorders. A number of such proteins have been described as required to mediate these activities.

Pro-inflammatory cells and cytokines that correlate with disease severity are present in the circulation of patients with neurodegenerative disease. Altered levels of inflammatory monocytes have been observed in the circulation of MND patients and in mouse models of MND (Butovsky O. et al. J. Clin. Invest. 122:3063 (2012).; Murdock BJ. , et al.Neurol.Neuroimmunol.Neuroinflammation 3:e242 (2016)). Furthermore, a small study involving treatment of MND patients with intravenous tocilizumab, a humanized antibody against IL-6R, showed that IV dosing reduced serum inflammatory cytokine levels and attenuated disease progression in a subpopulation of patients. (Fiala M., et al. Am. J. Neurodegener. Dis. 2:129-139 (2013)). Therapies targeting the inflammatory response associated with MND pathology have been investigated. However, rather than targeting the primary initiator that drives degeneration and disease, these are concerned with targeting IL-6 receptors that address effectors of inflammation. Due to CD14's role in initiation of the inflammatory cascade and production of pro-inflammatory mediators, inhibition or reduction of CD14-mediated inflammatory responses, both through mCD14 and directly through sCD14, It is proposed herein to provide a more effective treatment for inflammation associated with neurodegenerative diseases by addressing the cause rather than the effect of inflammation.

As described herein, inhibition or reduction of pro-inflammatory mediator production by cells located outside the CNS of patients with neurodegenerative disease using CD14 antagonist antibodies is associated with disease, such as those in the CNS. The onset or progression or symptoms thereof can be treated.

Accordingly, in one aspect, the present invention provides for the production of pro-inflammatory mediators (e.g., cytokines) in mammalian cells present in the periphery of a subject having or at risk of developing a neurodegenerative disease. Methods of inhibiting or reducing are provided. In one embodiment, these methods comprise contacting a peripheral cell comprising mCD14 with an anti-CD14 antagonist antibody in an amount sufficient to inhibit or reduce pro-inflammatory mediator production from the cell from Become or consist essentially of.

In another embodiment, the method comprises contacting peripherally circulating sCD14 with an anti-CD14 antagonist antibody in an amount sufficient to inhibit or reduce pro-inflammatory mediator production from peripheral cells. or essentially from.

Suitably the peripheral cells are immune cells (eg monocytes, macrophages, dendritic cells or T cells).

Proinflammatory mediators are typically cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1)-α, IL-6, IFN-γ, IFN-β, IL -1β, IL-8, IL-17, or IL-18.

In a related aspect, the methods defined above are used to treat neurodegenerative disease-mediated symptoms. These methods generally involve treating peripheral cells, including mCD14, or circulating sCD14 with a CD14 antagonist antibody, preferably an antibody sufficient to inhibit or reduce CD14-dependent production of pro-inflammatory mediators in peripheral cells. comprising, consisting of or consisting essentially of contacting with an amount of a CD14 antagonist antibody thereby treating a condition. In certain embodiments, the disease is MND or dementia. Suitably, when the disease is MND (eg, ALS), symptoms include, for example, progressive muscle atrophy, paralysis, spasticity, respiratory changes and hyperreflexia. In one embodiment, the disease is dementia and the symptoms include, for example, memory loss, depression, impaired communication, poor judgment, disorientation, confusion, sleep disturbance, motor symptoms, hallucinations, neuroleptic hypersensitivity. gender, behavioral changes and difficulties in speaking, swallowing and walking.

Yet another aspect of the invention provides a method of treating the development or progression of a neurodegenerative disease or a symptom thereof in a subject. These methods generally comprise, consist of, or consist essentially of systemically administering to a subject an effective amount of a CD14 antagonist antibody, thereby treating the development or progression of a neurodegenerative disease, or a symptom thereof, in a subject. . In some embodiments, production of one or more pro-inflammatory mediators in peripheral cells of the subject is inhibited or reduced. Suitably the pro-inflammatory mediator is a cytokine.

In a related aspect, the present invention provides one or more pro-inflammatory mediators (e.g., peripheral cell-mediated Use of CD14 antagonist antibodies for inhibiting or reducing the production of cytokines) or for treating the development or progression of neurodegenerative diseases is provided. In some embodiments, the CD14 antagonist is manufactured as a medicament for any one or more of those applications.

In some embodiments, the method identifies that the subject has or is at risk of developing or developing a neurodegenerative disease, preferably prior to administration of the CD14 antagonist antibody. further comprising: In instances of this type, the method comprises determining the presence of a neurodegenerative disease marker in a biological sample, preferably in a biological sample taken from the subject, preferably prior to administration of the CD14 antagonist antibody. , examples of biological samples include blood, serum, plasma, saliva, cerebrospinal fluid, urine, skin or other tissue, or small portions thereof.

In one embodiment, the disease is MND and the marker is, for example, SOD1, TDP-43, FUS, C9ORF72, ALS2, ALS4, ALS8, NEK1, UBQLN2, VCP, SETX, ANG, PFN1, MATR3, CHCHD10, selected from one or more of TUBA4A, TBK1, C21orf2 and OPTN or their expression products. In this embodiment, the presence of markers of neurodegenerative disease is preferably the presence or overexpression of marker gene expression products and/or marker genes (e.g. SOD1, TDP-43, FUS, C9ORF72, ALS2) in the biological sample. , ALS4, ALS8, NEK1, UBQLN2, VCP, SETX, ANG, PFN1, MATR3, CHCHD10, TUBA4A, TBK1, C21orf2 and OPTN mRNA or polypeptide). In some embodiments for MND, the presence of cytoplasmic deposition of TDP-43 positive inclusions and/or elevated serum and/or CSF levels of neurofilaments can also be determined.

In another embodiment, the disease is dementia such as Alzheimer's disease, FTD LBD (DLB and PDD) and vascular dementia, and the marker encodes amyloid precursor protein (APP) and presenilins 1 and 2 mutation in the gene, mutation in the ε4, 2 and 3 alleles of the apolipoprotein E (APOE) gene (APOE-ε4, APOE-ε2, APOE-ε3), Triggering receptor expressed on myeloid cells 2 (TREM2) gene, protein The MAPT gene on chromosome 17 that makes tau, the GRN gene, also called the PGRN gene, on chromosome 17 that makes the progranulin protein, chromosome 1 that produces a transactivation-responsive DNA-binding protein (TDP-43) with a molecular weight of 43 kDa. The TARDBP gene on chromosome 9, which encodes the valosin-containing protein, and the VCP gene on chromosome 3, which expresses charged multivesicular body protein 2B (also known as chromatin modifying protein 2B). selected from one or more of the mutations in the CHMP2B gene. Elevated serum and/or CSF levels of α-synuclein, S100A9 and S100B, chromogranin, circulating DNA, heat shock proteins and amyloid can also be determined.

In other embodiments, subjects at risk for neurodegenerative disease may also be tested for proinflammatory markers associated with disease in peripheral blood mononuclear cells (PBMCs), such as TNF-α, interleukin-1 (IL-1) - α, IL-6, IFN-γ, IFN-β, IL-1β, IL-8, IL-18, C-reactive protein (CRP), IL-17, chemokines, CD14-high monocytes and inflammatory mediators Identification can be made by determining the presence of elevated levels of one or more of the mRNA transcripts. In one embodiment, the disease is MND and the cytokines detected are selected from one or more of IL-6 or IL-17. In one embodiment, the disease is dementia and the cytokines detected are selected from IL-1, IL-6 and TNF-α.

In preferred embodiments, the CD14 antagonist antibody is IC14, or an antigen-binding fragment thereof.

A CD14 antagonist antibody can be administered alone or in combination with one or more adjuvant agents to treat the onset or progression of a neurodegenerative disease or symptoms thereof. Thus, in yet another aspect, the present invention provides pharmaceutical compositions, preferably formulated for systemic administration, for treating the development or progression of neurodegenerative diseases or symptoms thereof. These compositions comprise, consist or consist essentially of a CD14 antagonist antibody, optionally together with a pharmaceutically acceptable carrier or diluent. In one embodiment, the composition further comprises an adjunctive anti-neurodegenerative agent.

In a related aspect, the invention provides methods of treating the development or progression of a neurodegenerative disease or a symptom thereof in a subject. These methods generally involve concurrently administering to a subject an effective amount of a systemically administered CD14 antagonist antibody and an effective amount of an adjunctive anti-neurodegenerative agent, thereby causing development or progression of a neurodegenerative disease in the subject, or comprising, consisting of or consisting essentially of treating its symptoms. Preferably, the CD14 antagonist antibody and the adjunctive anti-neurodegenerative agent are administered in synergistically effective amounts.

In certain embodiments, the adjunctive anti-neurodegenerative agent is an anti-inflammatory agent. In one embodiment, the disease is MND (e.g., ALS) and the adjunctive anti-neurodegenerative agent is riluzole (Rilutek®/Teglutik®), a complement pathway inhibitor (e.g., PMX205 or eculizumab), agents that block the interaction of CD40 with CD40 ligands, such as antibodies that specifically bind CD40 and/or CD40 ligands (e.g., AT-1502), NurOwn stem cell therapy (BrainStorm Cell Therapeutics), selected from GM604, edaravone (Radicava®/Radicut®), macitinib, memantine or tiracemtib. In one embodiment, the disease is dementia such as LBD, AD or FTD and the adjunctive anti-neurodegenerative agent is a cholinesterase inhibitor. In another embodiment, the disease is AD and the adjuvant is an approved therapeutic agent selected from, for example, Aricept, Lazadaine, Namenda, Exelon and Namzaric.

In another related aspect, the invention provides the use of CD14 antagonist antibodies and adjunctive anti-neurodegenerative agents to treat the development or progression of a neurodegenerative disease, or symptoms thereof. In some embodiments, the CD14 antagonist antibody and adjunctive anti-neurodegenerative agent are manufactured as medicaments for systemic administration for the purposes of this application. Preferably, the CD14 antagonist antibody and the adjunctive anti-neurodegenerative agent are formulated for concurrent administration.

Embodiments of the present disclosure will now be described, by way of non-limiting example only, with reference to the following drawings.
Figure 1 shows the i.v. v. Levels of IL-17, neurofilament light chain (NFL) and IL-1β in CSF after administration are shown. Figure 2 shows the results of an in vitro assay evaluating the effect of IC14 on cytokine or chemokine levels following TDP-43 activation of primary human microglia. After pretreatment of primary human microglia with IC14 or control IgG4 antibody for 2 h, cells were treated with (A) mutant TDP-43 (mTDP43) or (B) wild-type TDP-43 (wtTDP43). Stimulated for 48 hours. Supernatants were harvested for multiplex cytokine analysis. Untreated cells received neither antibody nor TDP-43, reflecting basal levels of cytokine or chemokine production. Expression was measured as relative fluorescence units (RFU) and expressed as the mean and SEM of triplicate wells per sample. Same as description for FIG. 2A.

1. DEFINITIONS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

The articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element.

As used herein, "and/or" is interpreted selectively in addition to any and all possible combinations of one or more of the associated listed items. refers to and includes lack of combination when

Terms such as "concurrent administration" or "administering in parallel" or "administering in combination" refer to the administration of a single composition containing two or more agents or the administration of separate Each agent delivered as a composition and/or by separate routes is administered at the same time or at the same time or when all such agents are administered as a single composition. It refers to sequential administration within a short enough period of time to be equivalent. "Concurrently" means that the agents are administered at substantially the same time, preferably together in the same formulation. "Contemporaneously" means that the agents are administered at a close time, eg, one agent is administered within about one minute before or after another agent and within about one day. Any synchronization period is useful. Often, however, if not administered simultaneously, the agents will be administered within about 1 minute to about 8 hours, preferably within about 1 to about 4 hours. When administered at the same time, the agents are preferably administered at the same site on the subject. The term "same site" includes the exact same location, but can be within about 0.5 to about 15 centimeters, preferably within about 0.5 to about 5 centimeters. The term "separately" as used herein means that the agents are administered at intervals, eg, intervals of about one day to several weeks or months. Agents may be administered in either order. The term "sequentially" as used herein means that the agents are administered sequentially, eg, at intervals of minutes, hours, days or weeks. Where appropriate, the agents may be administered in regular repeating cycles.

The terms "ALS", "MND" and "Lou Gehrig's disease" may be used interchangeably herein to refer to the same condition. Both familial and sporadic ALS can be treated or their onset or progression delayed by the subject methods. All forms of ALS are contemplated herein.

The term "agonist" refers to a ligand that stimulates the receptor to which it binds. An agonist by the classical definition, whether an orthosteric agonist, an allosteric agonist, an inverse agonist or a co-agonist, binds to a receptor, alters the state of that receptor and results in a biological effect. properties and biological effects, whether direct or indirect, by activating chemical or physical signaling cascades to influence cell behavior, physical or biological resulting in a definable change in state of mind. As a result, agonism is defined as the property of an agonist to produce a biological effect. In the context of the present invention, CD14 agonists are e.g. by binding mCD14 on the surface of cells or sCD14 in circulation, such as damage-associated molecular pattern (DAMP) or pathogen-associated molecular pattern (PAMP) molecules and LPS. Any agonist that initiates a pro-inflammatory response is included. Non-limiting examples of DAMPs include SOD1, TDP-43, members of the heat shock protein family, as well as HMGB1, S100A, S100A9, S100B, α-synuclein, chromogranin, circulating DNA and RNA, amyloid and mutagenesis. Any other misfolded protein that initiates a pro-inflammatory response by binding to mCD14 on the surface of cells or to sCD14 in circulation is included. Non-limiting examples of PAMPs include LPS, other peptidoglycans and Gram-positive lipoteichoic acids, mycobacterial lipoarabinomannans, and viral envelope proteins.

The term "antagonist antibody" is used in its broadest sense and includes antibodies that inhibit or reduce the biological activity of the antigen (eg, CD14) to which it binds. For example, an antagonist antibody can partially or completely block the interaction of a receptor (eg, CD14) with a ligand (eg, DAMP or PAMP), or alter or downregulate the tertiary structure of the receptor. can substantially reduce interactions due to Thus, a CD14 antagonist antibody binds to CD14 and blocks, inhibits, abolishes, antagonizes, suppresses, reduces CD14 agonist activity to any significant degree (including those that do so significantly). or antibodies that reduce the effects of, for example, the Toll-like receptor (TLR) signaling pathway (e.g., TLR4 signaling pathway) and the TIR-domain-containing adapter-induced IFN-β (TRIF) pathway. activation of downstream pathways, or triggering of cellular responses (eg, production of pro-inflammatory mediators such as pro-inflammatory cytokines) to CD14 binding by CD14 ligands (eg, DAMPs or PAMPs).

The term "antibody" is used herein in the broadest sense, and specifically includes naturally occurring antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments , or any other antigen binding molecule so long as it exhibits the desired immunointeractivity. A naturally occurring "antibody" includes within its scope an immunoglobulin comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Heavy chain constant regions include specific CH domains (eg, CH1, CH2 and CH3). Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of antibodies may mediate binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system. Antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), subclass or variant thereof (e.g., L234A and IgG1 isotype with L235A double mutation (IgG1-LALA)). Antibodies can be of any species, chimeric, humanized or human. In other embodiments, the antibody is a homomeric heavy chain antibody (e.g., camelid antibody), which lacks the first constant region domain (CH1) but retains an otherwise intact heavy chain. and can bind to antigen through the antigen-binding domain. The heavy and light chain variable regions in antibody-modular recognition domain (MRD) fusions contain functional binding domains that interact with the antigen of interest.

As used herein, "variable domain" (light chain variable domain (VL), heavy chain variable domain (VH)) refers to the light and heavy chain domains directly involved in the binding of an antibody to antigen. Represents each of a pair of domains. Variable light and heavy chain domains have the same general structure, each containing four FRs whose sequences are widely conserved, connected by three CDRs or "hypervariable regions." FRs adopt a β-sheet conformation and CDRs can form loops that connect β-sheet structures. The CDRs in each chain are held in three-dimensional structure by the FRs and together with the CDRs from the other chain form the antigen binding site.

The term "antigen-binding portion" as used herein refers generally to the amino acid residues of an antibody that are responsible for antigen-binding, and generally includes the amino acid residues from the CDRs. Thus, "CDRs" or "complementarity determining regions" (also called "hypervariable regions") are the amino acid sequences of antibody light and heavy chains that form a three-dimensional loop structure that contributes to the formation of the antigen-binding site. are used interchangeably herein to refer to There are three CDRs in each variable region of the heavy and light chains, referred to as "CDR1," "CDR2," and "CDR3," for each variable region. As used herein, the term "CDR set" refers to a group of three CDRs present in a single variable region that binds antigen. The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) provides a well-defined sequence applicable to any variable region of an antibody. In addition to providing a residue numbering system, we also provide precise residue boundaries that define the three CDRs, which may be referred to as "Kabat CDRs". Lesk, 1987. J. Mol. These subdivisions are either 'L1', 'L2' and 'L3' or 'H1', 'H2'. , and “H3”, with “L” and “H” denoting the light and heavy chain regions, respectively.These regions are sometimes referred to as “Chothia CDRs” and have overlapping boundaries with the Kabat CDRs. Other boundaries defining CDRs that overlap the Kabat CDRs are described by Padlan (1995. FASEB J. 9: 133-139) and MacCallum (1996. J. Mol. Biol. 262(5): 732-745). Also, the boundary definition of other CDRs may not strictly follow one of these systems, but still overlap with the Kabat CDRs, provided that specific residues or groups of residues or even CDRs They may be shorter or longer, in light of predictions or experimental findings that overall does not significantly affect antigen binding.

As used herein, the term "framework region" or "FR" refers to the remaining sequences of a variable region minus the CDRs. Thus, antibody light and heavy chain variable domains comprise, from N-terminus to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. CDRs and FRs are typically described in Kabat, E.; A. , et al. , Sequences of Proteins of Immunological Interest, 5th ed. , Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or those residues from the "hypervariable loop".

As used herein, the terms "light chain variable region" ("VL") and "heavy chain variable region" (VH) refer to light and heavy chains, respectively, which have different primary amino acid sequences for each antibody. Refers to the region or domain of the N-terminal portion. The variable region of an antibody typically consists of the amino-terminal domains of light and heavy chains, which fold together to form a three-dimensional binding site for antigen. Based on structural similarity, a number of subtypes of VH and VL have been defined, eg, as described in the Kabat database.

The term "chimeric antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species and constant region sequences from another species, e.g., murine heavy and light chain variable regions linked to human constant regions. Refers to an antibody having a region.

“Humanized” forms of non-human (eg, rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, a humanized antibody is a non-human species such as mouse, rat, rabbit or non-human primate ( A human immunoglobulin (recipient antibody) that has been replaced with residues from the hypervariable regions of the donor antibody). In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Thus, the FRs and CDRs of the humanized antibody need not correspond exactly to the parental (i.e., donor) sequence, e.g., the CDRs or consensus framework of the donor antibody must contain at least one amino acid residue substitution, insertion, and/or mutagenized by deletion so that the CDRs or FRs at that site do not correspond to either the donor antibody or the consensus framework. Typically, however, such mutations are not extensive and generally avoid "key residues" involved in antigen binding. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region within a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (e.g., Winnaker, From See Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). A "consensus immunoglobulin sequence" may thus comprise a "consensus framework region" and/or a "consensus CDR". In a family of immunoglobulins, each position in the consensus sequence is occupied by the most frequently occurring amino acid at that position in that family. If two amino acids occur with the same frequency, either can be included in the consensus sequence. Generally, a humanized antibody comprises at least one, and typically two, substantially all of the variable domains in which all or substantially all of the hypervariable loops are those of a non-human immunoglobulin. Corresponding and all or substantially all of the FRs are of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details see Jones et al. (1986. Nature 321:522-525), Riechmann et al. (1988. Nature 332:323-329) and Presta (1992. Curr. Op. Struct. Biol. 2:593-596). A humanized antibody may be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. A humanized antibody may contain sequences from more than one class or isotype, and specific constant domains are engineered to optimize the desired effector functions using techniques well known in the art. can be selected. As used herein, the term "key residue" refers to certain residues within the variable region that have a greater impact on the binding specificity and/or affinity of an antibody, particularly a humanized antibody. refers to the residue of Key residues include: residues flanking the CDRs, potential glycosylation sites (which can be either N- or O-glycosylation sites), rare residues, antigen-interacting residues that can interact with CDRs, canonical residues, contact residues between the heavy and light chain variable regions, residues within the Vernier zone, and variable heavy Including, but not limited to, one or more of the residues in the region of overlap between the Chothia definition of chain CDR1 and the Kabat definition of the first heavy chain framework.

As used herein, the "Vernier" zone is the alignment of the CDR structure to an antigen, as described by Foote and Winter (1992. J. Mol. Biol. 224: 487-499). Refers to a subset of framework residues that can be fine-tuned for fit. The Vernier zone residues form the underlying layer of the CDRs and can influence the structure of the CDRs and the affinity of the antibody.

As used herein, the term "canonical" residue is defined by Chothia et al. (1987. J. Mol. Biol. 196: 901-917; 1992. J. Mol. Biol. 227: 799-817; both of which are incorporated herein by reference). Refers to the residues in the CDRs or frameworks that define the canonical CDR structure of. According to Chothia et al., the essential parts of the CDRs of many antibodies have nearly identical peptide backbone confirmations, despite great diversity at the level of amino acid sequence. Each canonical structure primarily specifies a set of peptide backbone torsion angles for successive segments of loop-forming amino acid residues.

As used herein, the terms "donor" and "donor antibody" refer to an antibody that contributes one or more CDRs to an "acceptor antibody." In some embodiments, the donor antibody is an antibody from a different species than the antibody from which the FRs are obtained or derived. In the context of humanized antibodies, the term "donor antibody" refers to a non-human antibody that contributes one or more CDRs.

As used herein, the terms "acceptor" and "acceptor antibody" refer to at least 80%, at least 85%, at least 90%, at least 95%, at least 98% of one or more amino acid sequences of an FR. , or antibodies that provide 100%. In some embodiments, the term "acceptor" refers to the antibody amino acid sequences that provide the constant regions. In other embodiments, the term "acceptor" refers to antibody amino acid sequences that provide for one or more of the FR and constant regions. In certain embodiments, the term "acceptor" refers to at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or 100% of one or more amino acid sequences of the FR. Refers to the human antibody amino acid sequences provided. According to this embodiment, the acceptor may contain at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 amino acid residues that are not present at one or more specific positions of a human antibody. . Acceptor framework regions and/or acceptor constant regions are, for example, germline antibody genes, mature antibody genes, functional antibodies (e.g., antibodies known in the art, antibodies under development, or commercially available antibodies). may be derived from or obtained therefrom.

The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may be produced by amino acid residues not encoded by human germline immunoglobulin sequences, e.g., in the CDRs, particularly CDR3, e.g. mutations introduced by somatic mutation). However, the term "human antibody" as used herein includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. is not intended.

The terms "heavy chain variable region CDR1" and "H-CDR1" are used interchangeably, the terms "heavy chain variable region CDR2" and "H-CDR2", "heavy chain variable region CDR3" and "H-CDR3". , the terms "light chain variable region CDR1" and "L-CDR1", the terms "light chain variable region CDR2" and "L-CDR2" and the "light chain variable region CDR3" and "L-CDR3" antibodies The same applies to the term fragment. Throughout this specification, complementarity determining regions (“CDRs”) are defined according to the Kabat definition unless otherwise specified. The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDR regions (Kabat et al., (1991), 5th edition, NIH publication No. 91-3242).

Antigen binding can be performed by "fragments" or "antigen-binding fragments" of intact antibodies. Both terms are used interchangeably herein. Examples of binding fragments encompassed by the term "antibody fragment" of an antibody are Fab fragments, which are monovalent fragments consisting of the VL, VH, CL and CH1 domains; two Fab fragments linked by a disulfide bridge at the hinge region. Fd fragment consisting of VH and CH1 domains; Fv fragment consisting of VL and VH domains of a single arm of the antibody; single domain antibody (dAb) consisting of VH domain fragments (Ward et al., 1989. Nature 341:544-546); as well as isolated complementarity determining regions (CDRs).

A "single-chain variable fragment (scFv)" is a monovalent molecule in which the VL and VH regions pair together (known as a single-chain Fv (scFv); e.g., Bird et al., 1988. Science 242:423-426; and Huston et al., 1988. Proc. Natl. Acad. Sci. Although the two domains VL and VH are encoded by separate genes, they are linked by an artificial peptide linker that allows them to be combined into a single protein chain using recombinant methods. can be done. Such single chain antibodies comprise one or more antigen binding portions. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

The term "monoclonal antibody" and the abbreviations "MAb" and "mAb" as used herein refer to antibodies obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies that make up the population The antibodies are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody can be produced, for example, by a single clone of antibody-producing cells such as a hybridoma. The term "hybridoma" generally refers to the product of cell fusion between a cultured neoplastic lymphocyte and a primed B or T lymphocyte that exhibits the specific immune potential of the parental cell.

An antibody that "binds" an antigen of interest (e.g., CD14) binds the antigen with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting cells or tissues that express the antigen, and does not bind other antigens. An antibody that does not significantly cross-react with the protein. In such embodiments, the extent of antibody binding to the "non-target" protein is determined, for example, by fluorescence-activated cell sorting (FACS) analysis, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation or radioimmunoprecipitation (RIA). ), less than about 10% of the antibody, oligopeptide or other organic molecule binds to its specific target protein. Antibodies that antagonize CD14 to which it binds therefore preferably inhibit or reduce the production of pro-inflammatory mediators such as pro-inflammatory cytokines/chemokines. With respect to the binding of an antibody to a target molecule, the terms "specific binding" to or "specifically binds" or "specific" to a particular polypeptide or epitope on a particular polypeptide target refer to means a different binding than a non-specific interaction. Specific binding can be measured, for example, by determining the binding of a molecule relative to the binding of a control molecule, which is a molecule of similar structure that generally lacks binding activity. For example, specific binding can be determined by competition of a target, eg, an excess of unlabeled target, with a similar control molecule. In this case, specific binding is indicated when binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. The specific region of an antigen to which an antibody binds is typically called the "epitope." The term "epitope" broadly includes sites on an antigen that are specifically recognized by an antibody or T-cell receptor or otherwise interact with a molecule. Epitopes are generally those active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally can have specific three dimensional structural characteristics, as well as specific charge characteristics. As will be appreciated by those of skill in the art, virtually anything to which an antibody can specifically bind can be an epitope.

The term "cell" as used herein refers to any mammalian cell outside the CNS that contains mCD14 and produces pro-inflammatory mediators (eg, cytokines). Non-limiting examples are immune cells, epithelial cells, osteoblasts, fibroblasts, and smooth muscle cells.

Throughout this specification, unless the context requires otherwise, the terms "comprise," "comprises," and "comprising" may be used to refer to a recited step or element or step or It is understood to include groups of elements, but not to exclude any other steps or elements or groups of steps or elements. Thus, use of terms such as "comprising" indicates that while the listed element is required or mandatory, other elements are optional and may or may not be present. point to "Consisting of" means including and limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed element is required or required and that other elements cannot be present. The term “consisting essentially of” includes any elements listed after the phrase and is intended to be limited to other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the listed elements. means. Thus, the phrase "consisting essentially of" means that the listed element is required or essential but that other elements are optional and whether or not they affect the activity or action of the listed element. indicates that it may or may not be present depending on the

As used herein, reference to "dementia" refers to any disease belonging to a syndrome in which cognitive function (ie, the ability to process thoughts) deteriorates beyond what can be expected from normal aging. Without limiting the invention to any one disease, examples of dementia include AD, dementia with Lewy bodies, ie dementia with Lewy bodies (DLB) and Parkinson's disease dementia, vascular and frontotemporal dementia (FTD).

An "effective amount," in the context of treating a condition, is a dose, either as a single dose or as part of a series, of the condition that causes symptoms in an individual in need of such treatment or prevention. It means administration of an amount of an agent or composition effective for prophylaxis, prevention of such symptoms, and/or treatment of existing symptoms. The effective amount will depend on the age, health and physical condition of the individual being treated and whether symptoms of disease are present, the taxonomy of the individual being treated, the formulation of the composition, the assessment of the medical condition, and other relevant factors. Varies accordingly. Optimal dosing schedules can be calculated from measurements of drug accumulation in a subject's body. Optimum dosages may vary according to relative potency in individual subjects, and can generally be estimated based on EC50 values found to be effective in in vitro and in vivo animal models. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. It is expected that the amount will fall within a relatively broad range that can be determined through routine trials.

"Hybridization" is used herein to describe the pairing of complementary nucleotide sequences to produce a DNA-DNA or DNA-RNA hybrid. Complementary base sequences are sequences that are related by the rules of base pairing. In DNA, A pairs with T and C with G. In RNA, U pairs with A and C pairs with G. In this regard, the terms "match" and "mismatch" as used herein refer to the hybridization potential of pairing nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical AT and GC base pairs described above. Mismatches are other combinations of nucleotides that do not hybridize efficiently. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which is a Watson-Crick, Hoogsteen or inverted Hoogsteen hydrogen bond between complementary nucleoside or nucleotide bases (nucleobases) of a strand of an oligomeric compound. can be a bond. For example, adenine and thymine are complementary nucleobases and pair through the formation of hydrogen bonds. Hybridization can occur under various circumstances as known to those of skill in the art.

As used herein, the term "immune cell" refers to cells belonging to the immune system that reside outside the CNS. Immune cells include cells of hematopoietic origin, including but not limited to T lymphocytes (T cells), B lymphocytes (B cells), Natural Killer (NK) cells, granulocytes, neutrophils, macrophages, Monocytes, dendritic cells, and specialized forms of any of the above, such as plasmacytic dendritic cells, Langerhans cells, plasma cells, natural killer T (NKT) cells, T helper cells, and cytotoxic T lymphocytes A sphere (CTL) is included.

Reference herein to "immunointeractive" and its grammatical equivalents means any interaction between molecules, especially when one of the molecules is or mimics a component of the immune system. , reactions, or other forms of association.

As used herein, "inhibit", "inhibiting", "decrease" or "decreasing" with respect to "pro-inflammatory mediator production" by cells, etc. The term refers to at least a small but measurable reduction in the level or amount of proinflammatory mediators produced by peripheral cells. In embodiments, the production of proinflammatory mediators by the cells is inhibited or reduced by at least 20% relative to untreated controls, in further embodiments the inhibition or reduction is by at least 50%, and in still further embodiments , the inhibition or reduction is at least 70%, in embodiments the inhibition or reduction is at least 80%. Such reduction in production of pro-inflammatory mediators can reduce detrimental effects of the inflammatory mediator cascade in in vivo embodiments.

Suitable in vitro assays (eg, ELISA, RT-PCR) can be used to assess the effectiveness of CD14 antagonist antibodies in inhibiting or reducing pro-inflammatory mediator production by peripheral cells. For example, competitive RT-PCR techniques can be used to measure levels of cytokine mRNA obtained from cells, and levels of expressed cytokines released from cells can be measured, for example, by specific can be measured by a sandwich ELISA using one or more monoclonal antibodies that bind to In vivo screening can also be performed by following procedures well known in the art. For example, a CD14 antagonist antibody is administered to an animal model (eg, mouse) and blood is collected to assess levels of various cytokines. Those skilled in the art are well familiar with the techniques available for measuring cytokine production. Based on the results, an appropriate dosage range and systemic administration route can also be determined.

"Isolated" means material that is substantially or essentially free from components that normally accompany it in its natural state.

The term "ligand" as used herein refers to any molecule capable of binding to a receptor.

As used herein, the phrase "motor neuron disease (MND)" refers to neurological disorders that selectively destroy motor neurons.

The phrase "neurodegenerative disease" means a disease characterized by progressive nervous system dysfunction. Neurodegenerative diseases comprise a heterogeneous group of diseases of the central or peripheral nervous system with many different etiologies. Such conditions may be, but are not limited to, hereditary, toxic or secondary to metabolic processes, and may result from infection. Neurodegenerative conditions are progressive conditions that can be age-related or chronic. Such conditions may be characterized by abnormalities in relatively specific regions of the brain or specific populations of neurons. The specific cell populations affected in different neurodegenerative conditions typically determine the clinical phenotype of the condition. In particular, neurodegenerative conditions can be associated with atrophy of specific affected central or peripheral nervous system structures.

Exemplary neurodegenerative diseases or conditions include amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive Motor neuron diseases (MND), such as bulbar palsy (PBP) and pseudobulbar palsy, include, but are not limited to. Other exemplary neurodegenerative diseases or conditions include Alzheimer's disease, Parkinson's disease-like syndrome, dementia with Lewy bodies (DLB and PDD), vascular dementia, frontotemporal dementia, mesolimbic cognition. familial dementia with spastic paraplegia and dementia such as AIDS-related dementia.

Other exemplary neurodegenerative diseases include Parkinson's disease, spinal muscular atrophy, hereditary spinal muscular atrophy, Charcot-Marie-Tooth disorder, Kennedy disorder and post-polio syndrome, multiple sclerosis, diffuse cortical atrophy. , Pick's disease, thalamic degeneration, Huntington's chorea, corticostriatal spinal cord degeneration, corticobasal ganglion degeneration, cerebral cerebellar degeneration, polyglucosan body disease, Shy-Drager syndrome, olivopontocerebellar atrophy, progression Supranuclear palsy, muscular dystonia degenerative, Hallerholden-Spatz disease, Mage syndrome, familial tremor, Gilles de la Tourette syndrome, acanthrocytic chorea, Friedreich's ataxia, Holmes-type familial corticocerebellum Atrophy, Gerstmann-Straussler-Scheinker disease, hereditary muscular atrophy, spastic paraplegia, peroneal muscular atrophy, hypertrophic interstitial polyneuropathy, polyneuritic hereditary ataxia, and optic neuropathy. Those skilled in the art will appreciate these and other mild, moderate or severe neurodegenerative conditions whose underlying inflammatory component contributing to disease pathology can be treated according to the methods of the present invention.

It is common for people to have mixed dementia, a combination of two or more diseases, at least one of which is dementia. For example, some people have both Alzheimer's disease and vascular dementia, and a few people with FTD also have motor neuron disease (FTD/MND) (FTD with amyotrophic lateral sclerosis). or FTD/ALS). Thus, reference to a "neurodegenerative disease" as used herein should be a reference to a subject diagnosed as having or at risk of developing one or more neurodegenerative diseases. should be understood.

The terms "patient," "subject," or "individual," as used interchangeably herein, are those who have been diagnosed with a neurodegenerative disease or have an increased likelihood of developing a neurodegenerative disease. It refers to any identified subject, particularly a vertebrate subject, and more particularly a mammalian subject. Patients may be healthy or may show prior signs of neurodegenerative disease, such as muscle wasting or memory loss, which are not related to: Alternatively, the subject may have a genetic predisposition to the disease.

A "subject" is generally a human subject, but treatment of the onset or progression of neurodegenerative diseases and conditions also includes treatment of horses for equine motor neuron disease, treatment of dogs for canine spinal muscular atrophy and animal studies. It can be important in trials and the like. Thus, references herein to "patient", "subject" or "individual" include both human and non-human mammals, including but not limited to companion animals such as horses, dogs and cats, and Including laboratory animals such as mice, rats, guinea pigs, hamsters, rabbits, pigs and non-human primates.

As used herein, the terms "systemic administration" or "administered systemically" or "systemically administered" refer to administering an agent into a subject outside the central nervous system. means to introduce Systemic administration includes any route of administration other than administration directly to the spine or brain. As such, intrathecal and epidural administration as well as intracranial injection or implant insertion are clearly not within the scope of the term "systemically administered" or "systemically administered". It will be appreciated that systemic administration does not preclude therapeutic effects from occurring in the CNS.

Pharmaceutical compositions useful in the present invention may be administered orally in any acceptable form such as, for example, tablets, liquids, capsules, or powders; by intravenous, intraperitoneal, intramuscular, subcutaneous or parenteral injection; Systemic administration may be by skin diffusion or electrophoresis; and by minipumps or other implantable sustained release devices or formulations. According to some embodiments, systemic administration is performed by a route selected from the group consisting of intraperitoneal, intravenous, subcutaneous and intranasal administration, and combinations thereof.

References to "peripheral" as used herein include cells expressing mCD14, such as the circulatory system (e.g., cardiovascular and lymphatic systems) and the peripheral nervous system, or the body where circulating sCD14 is found. (not part of the CNS).

"Pharmaceutically acceptable carrier" means a pharmaceutical carrier comprising materials that are not biologically or otherwise undesirable, i.e., materials that do not cause any or substantial adverse reactions. It can be administered to the subject along with the active agent of choice. Carriers may include excipients and other additives such as diluents, surfactants, coloring agents, wetting agents, emulsifying agents, pH buffering agents, preservatives, and transfection agents.

Similarly, "pharmacologically acceptable" salts, esters, amides, prodrugs or derivatives of the compounds provided herein are not biologically or otherwise undesirable salts, esters, , amides, prodrugs or derivatives.

The terms "polynucleotide", "genetic material", "genetic form", "nucleic acid" and "nucleotide sequence" refer to RNA, cDNA, genomic DNA, synthetic forms and mixed polymers as readily understood by those of ordinary skill in the art. , including both sense and antisense strands, may be chemically or biochemically modified, or may contain non-natural or derivative nucleotide bases.

The term "proinflammatory mediator" means an immunomodulatory agent that favors inflammation. Such agents include chemokines, interleukins (ILs), lymphokines, and cytokines such as tumor necrosis factor (TNF), as well as growth factors. In certain embodiments, a proinflammatory mediator is a "proinflammatory cytokine." Typically, proinflammatory cytokines include IL-1α, IL-1β, IL-6, and TNF-α, which are generally involved in early responses. Other pro-inflammatory mediators include LIF, IFN-γ, IFN-β, IFN-α, OSM, CNTF, TGF-β, GM-CSF, TWEAK, IL-11, IL-12, IL-15, IL -17, IL-18, IL-19, IL-20, IL-8, IL-16, IL-22, IL-23, IL-31 and IL-32 (Tato et al., 2008. Cell 132:900; Cell 132:500, Cell 132:324). Pro-inflammatory mediators can act as endogenous pyrogens (IL-1, IL-6, IL-17, TNF-α), macrophages and mesenchymal cells such as fibroblasts, epithelial and endothelial cells ), may stimulate the production of acute phase proteins, or may attract inflammatory cells. In certain embodiments, the term "pro-inflammatory cytokine" relates to TNF-α, IL-1α, IL-6, IFN-β, IL-1β, IL-8, IL-17 and IL-18.

The term "receptor" refers to a cell-associated protein that binds a biologically active molecule called a "ligand." This interaction mediates the effect of the ligand on the cell. In general, binding of a ligand to a receptor results in a conformational change in the receptor that causes the receptor to interact with other molecules on the surface or inside the cell, which in turn causes the cell to lead to changes in the metabolism of Metabolic events often linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increased cyclic AMP production, intracellular calcium mobilization, membrane lipid mobilization, cell adhesion, Inositol lipid hydrolysis, phospholipid hydrolysis and modulation of cellular pathways (eg, stimulation or inhibition of production of one or more pro-inflammatory mediators) are included.

The term "sequence identity" as used herein refers to the extent to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over the window of comparison. Thus, "percentage of sequence identity" refers to comparing two optimally aligned sequences over a window of comparison, identical nucleobases (e.g., A, T, C, G, I) or identical amino acid residues. Groups such as Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met are present in both sequences determining the number of positions matched to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to determine sequence identity. Calculated by taking the percentage of gender. For the purposes of the present invention, "sequence identity" is understood to mean a "match percentage" calculated by an appropriate method. For example, sequence identity analysis can be performed using the DNASIS computer program (version 2.5 for windows; Hitachi Software engineering Co., Ltd., South San Francisco) using standard defaults as used in the reference manual accompanying the software. available from Francisco, California, USA).

"Similarity" refers to the percentage of the number of amino acids that are identical or constitute conservative substitutions as defined in Table 1.

Similarity can be determined using a sequence comparison program such as GAP (Deveraux et al., 1984. Nucleic Acids Res. 12, 387-395). Thus, sequences of similar or substantially different lengths to those described herein can be compared by inserting gaps into the alignment, such gaps being e.g. , determined by the comparison algorithm used by GAP.

Terms used to describe the sequence relationship between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "sequence percentage of sex" and "substantial identity". A “reference sequence” is at least 12, but frequently 15-18, and often at least 25, monomeric units in length, including nucleotides and amino acid residues. Since the two polynucleotides may contain (1) similar sequences (i.e., only a portion of the complete polynucleotide sequence) between the two polynucleotides, and (2) sequences that differ between the two polynucleotides, respectively, Sequence comparison between two (or more than two) polynucleotides typically compares the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. It is done by A "comparison window" is at least 6, usually about 50 to about 100, more usually about 50 to about 100, in which the sequences are compared to the same number of contiguous positions of a reference sequence after the two sequences are optimally aligned. Refers to a conceptual segment of 100 to about 150 contiguous positions. The comparison window can include additions or deletions (ie, gaps) of about 20% or less compared to the reference sequence (not including additions or deletions) for optimal alignment of the two sequences. The optimal alignment of sequences for aligning comparison windows was determined by computer-implemented algorithms (GAP, BESTFIT, FASTA, included in Wisconsin Genetics Software Package Release 7.0, Genetic Computer Group, 575 Science Drive Madison, WI, USA). , and TFASTA) or by any of a variety of methods tested and selected (i.e., resulting in the highest percentage of homology over the comparison window). For example, Altschul et al. , 1997. Nucleic Acids Res. 25:3389, also the BLAST family of programs. A detailed discussion of sequence analysis can be found in Ausubel et al. , "Current Protocols in Molecular Biology," John Wiley & Sons Inc, 1994-1998, Chapter 15, Unit 19.3.

As used herein, "stringency" refers to the temperature and ionic strength conditions and the presence or absence of certain organic solvents during hybridization. The higher the stringency, the higher the degree of complementarity observed between the sequences. As used herein, "stringent conditions" refer to temperature and ionic conditions under which only polynucleotides having a high proportion of complementary bases, preferably exact complementarity, will hybridize. The stringency required is nucleotide sequence dependent, depends on various components present during hybridization, and varies greatly when nucleotide analogs are used. Generally, stringent conditions are selected to be about 10°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a complementary probe. It will be appreciated that a polynucleotide will hybridize to a target sequence under at least low stringency conditions, preferably at least intermediate stringency conditions, and more preferably high stringency conditions. References herein to low stringency conditions include at least about 1% v/v to at least about 15% v/v formamide and at least about 1 M to at least about 2 M salt for hybridization at 42°C; and includes and includes at least about 1M to at least about 2M salt for washing at 42°C. Low stringency conditions also included 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65°C, and at room temperature. Washing can include (i) 2×SSC, 0.1% SDS, or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS. Intermediate stringency conditions are at least about 16% v/v to at least about 30% v/v formamide and at least about 0.5M to at least about 0.9M salt for hybridization at 42°C, and Contains and includes at least about 0.5M to at least about 0.9M salt for washing at 42°C. Intermediate stringency conditions are also 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS, and 42% for hybridization at 65°C. (i) 2×SSC, 0.1% SDS, or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at °C. can contain. High stringency conditions include at least about 31% v/v to at least about 50% v/v formamide and at least about 0.01 M to at least about 0.15 M salt for hybridization at 42°C and contains and includes at least about 0.01 M to at least about 0.15 M salt for washing with. High stringency conditions also include 1% BSA, 1 mM EDTA, 0.5M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65°C, and washing at temperatures above 65°C. For . One embodiment of high stringency conditions is hybridization in 6xSSC at about 45°C, followed by one or more cycles in 0.2xSSC, 0.1% SDS at 65°C. Including washing. One embodiment of very high stringency conditions is hybridization in 0.5 M sodium phosphate, 7% SDS at 65° C., followed by hybridization in 0.2×SSC, 1% SDS at 65° C. Including one or more washes. Other stringent conditions are well known in the art. Those skilled in the art will recognize that various factors can be manipulated to optimize specificity of hybridization. Optimization of the stringency of the final wash can help ensure a high degree of hybridization. For detailed examples, see pages 2.10.1-2.10.16 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra, and MOLECULAR CLONING. See Sections 1.101-1.104 of A LABORATORY MANUAL (Sambrook et al., eds.) (Cold Spring Harbor Press 1989).

As used herein, references to "symptoms" of neurodegenerative disease are physical or mental characteristics that are considered indicative of disease. Non-limiting MND-mediated disease symptoms include progressive muscle wasting, paralysis, spasticity, hyperreflexia, and other symptoms such as dysphagia, leg weakness, slurred speech, gait disturbance, facial Weakness, breathing changes and muscle spasms are included. Non-limiting dementia disease symptoms include recent speech, difficulty remembering names or events, apathy and depression (early symptoms), communication impairment, poor judgment, disorientation, confusion, behavioral changes and speech, Difficulty swallowing and walking (late symptoms) include. Examples of other symptoms that may be indicative of or associated with neurodegenerative disease include, for example, hallucinations, sleep disturbances, motor symptoms and neuroleptic hypersensitivity. Subjects typically exhibit one or several symptoms, depending on the disease and the individual subject. Determining which symptoms are considered indicative of a particular neurodegenerative disease is well within the skill of the art.

As used herein, terms such as "treatment" and "treating" refer to a subject in need of treatment, i.e., having or being diagnosed with a neurodegenerative disease, or obtaining a desired pharmacological and/or physiological effect in a subject at risk of developing a neurodegenerative disease. "Treatment"
(a) delaying the onset and/or progression of a neurodegenerative disease;
(b) ameliorating symptoms of a neurodegenerative disease;
(c) inhibiting a neurodegenerative disease or a symptom thereof; and/or (d) improving or prolonging quality of life. References to "treatment,""treat," or "treating" do not necessarily mean curing a subject or preventing disease progression indefinitely. Although the subject may eventually succumb to the neurodegenerative disease, the onset of the disease or condition is delayed, thus prolonging quality of life for a longer period of time than without treatment.

Indications of successful "treatment" include any objective or subjective parameter such as abatement; remission; a decline in memory or condition that is more acceptable to the patient; making the exacerbation end point less debilitating; or improving the subject's physical or mental health. Treatment or amelioration of symptoms may be based on objective or subjective parameters, such as the results of physical examination, neurological examination, and/or psychiatric evaluation.

As used herein, the application of underlining or italics to the name of a gene refers to the gene as opposed to its protein product, which is denoted by the name of the gene without any underlining or italics. pointed out. For example, "CD14" refers to the CD14 gene, while "CD14" refers to the protein products produced from transcription and translation and/or alternative splicing of the "CD14" gene.

Each embodiment described herein applies to each and every embodiment mutatis mutandis unless specifically stated otherwise.

2. Compositions and Methods for Treating the Development or Progression of Neurodegenerative Diseases or Symptoms Thereof The present invention provides methods and compositions comprising CD14 antagonist antibodies for treating the development or progression of neurodegenerative diseases or symptoms thereof. do.

2.1 CD14 Antagonist Antibodies The present invention provides antibodies that bind CD14 (e.g., mCD14 or sCD14) and block the binding of DAMPs or PAMPs to CD14 and/or bind CD14 to produce pro-inflammatory cytokines. Any CD14 antagonist antibody that inhibits or reduces a CD14 agonist-mediated response that results in the production of pro-inflammatory mediators such as CD14 is envisioned. In some embodiments, a CD14 antagonist antibody of the invention inhibits binding of a CD14 agonist, preferably DAMP or PAMP, to CD14, thus inhibiting or reducing pro-inflammatory cytokine production. In an example of this type, the CD14 antagonist antibody is the 3C10 antibody (van Voohris et al., 1983. J. Exp. Med. 158. : 126-145; Juan et al., 1995. J. Biol. 18 antibody (Bazil et al., 1986. Eur. J. Immunol. 16(12): 1583-1589; Juan et al., 1995. J. Biol. Chem. 270(10): 5219-5224), 4C1 antibody (Adachi et al., 1999. J. Endotoxin Res. 5: 139-146; Tasaka et al., 2003. Am. J. Respir. Cell. Mol. Biol.; 2003. 29(2): 252-258) In addition, the 28C5 and 23G4 antibodies inhibit LPS binding and pro-inflammatory cytokine production, and the 18E12 antibody partially inhibits LPS binding and inhibits pro-inflammatory cytokine production (Leturcq et al. al., US Pat. Nos. 5,820,858, 6,444,206 and 7,326,569). In some embodiments, the CD14 antagonist antibodies of the invention inhibit binding of CD14 to a TLR, such as TLR4, thereby blocking a CD14 agonist-mediated response, examples of which include WO2002/ Examples include the F1024 antibody disclosed in 42333 pamphlet. Each of the above references relating to CD14 antagonist antibodies is hereby incorporated by reference in its entirety. The CD14 antagonist antibody can be a full-length immunoglobulin antibody or an antigen-binding fragment of an intact antibody, representative examples of which are Fab fragments, F(ab')2 fragments, Fd fragments consisting of VH and CH1 domains. , an Fv fragment consisting of the VL and VH domains of a single arm of the antibody, a single domain antibody (dAb) fragment consisting of the VH domain (Ward et al., 1989. Nature 341:544-546), and isolated CDRs. is mentioned. Suitably the CD14 antagonist antibody is a chimeric, humanized or human antibody.

In some embodiments, the CD14 antagonist antibody is selected from the antibodies disclosed in US Pat. No. 5,820,858:

(1) Array:
QSPASLAVSLGQRATISC RASESVDSFGNSFMH WYQQKAGQPPKSSIY RAANLES GIPARFSGSGSRTDFTLTINPVEADDVATYFC QQSYEDPWT FGGGTKLGNQ [SEQ ID NO: 1]
a VL domain (3C10 VL) comprising, consisting of or consisting essentially of; S [SEQ ID NO: 2]
a VH domain (3C10 VH) comprising, consisting of or consisting essentially of
an antibody comprising;

(2) Sequence: QSPASLAVSLGQRATISC RASESVDSYVNSFLH WYQQKPGQPPKLLIY RASNLQS GIPARFSGSGSRTDFTLTINPVEADDVATYCC QQSNEDPTT FGGGTKLEIK [SEQ ID NO: 3]
a VL domain (28C5 VL) comprising, consisting of or consisting essentially of; LVTVSA [SEQ ID NO: 4]
a VH domain comprising, consisting of or consisting essentially of (28C5 VH)
antibodies, including

(3) Sequence: QTPSSLSASLGDRVTISC RASQDIKNYLN WYQQPGGTVKVLIY YTSRLHS GVPSRFSGSGSGTDYSLTISNLEQEDFATYFC QRGDTLPWT FGGGTKLEIK [SEQ ID NO: 5]
a VL domain (18E12 VL) comprising, consisting of or consisting essentially of; NFDY WGQGTTLTVSS [SEQ ID NO: 6]
a VH domain comprising, consisting of or consisting essentially of (18E12 VH)
Antibodies, including

Antibodies comprising the VL and VH CDR sequences of the above antibodies are also envisioned, representative embodiments of which include:

(1) a) comprising L-CDR1, L-CDR2 and L-CDR3, where L-CDR1 comprises the sequence RASESVDSFGNSFMH [SEQ ID NO: 7] (3C10 L-CDR1); L-CDR2 comprises the sequence RAANLES [SEQ ID NO: 8] (3C10 L-CDR2); and L-CDR3 comprises the sequence QQSYEDPWT [SEQ ID NO: 9] (3C10 L-CDR3); and b) H-CDR1, H- comprising CDR2 and H-CDR3, with H-CDR1 comprising the sequence SYAMS [SEQ ID NO: 10] (3C10 H-CDR1); H-CDR2 comprising the sequence SISSGGTTYYPDNVKG [SEQ ID NO: 11] (3C10 H-CDR2); and an antibody comprising an antibody VH domain, or an antigen-binding fragment thereof, wherein H-CDR3 comprises the sequence GYYDYHY [SEQ ID NO: 12] (3C10 H-CDR3);

(2) a) comprising L-CDR1, L-CDR2 and L-CDR3, where L-CDR1 comprises the sequence RASESVDSYVNSFLH [SEQ ID NO:13] (28C5 L-CDR1); L-CDR2 comprises the sequence RASNLQS [SEQ ID NO:14] (28C5 L-CDR2); and L-CDR3 comprises the sequence QQSNEDPTT [SEQ ID NO: 15] (28C5 L-CDR3); and b) H-CDR1, H- comprising CDR2 and H-CDR3, with H-CDR1 comprising the sequence SDSAWN [SEQ ID NO: 16] (28C5 H-CDR1); H-CDR2 comprising the sequence YISYSGSTSYNPSLKS [SEQ ID NO: 17] (28C5 H-CDR2); and an antibody comprising an antibody VH domain, or an antigen-binding fragment thereof, wherein H-CDR3 comprises the sequence GLRFAY [SEQ ID NO: 18] (28C5 H-CDR3); and

(3) a) comprising L-CDR1, L-CDR2 and L-CDR3, wherein L-CDR1 comprises the sequence RASQDIKNYLN [SEQ ID NO:19] (18E12 L-CDR1); L-CDR2 comprises the sequence YTSRLHS [SEQ ID NO:20] (18E12 L-CDR2); and L-CDR3 comprises the sequence QRGDTLPWT [SEQ ID NO: 21] (18E12 L-CDR3); and b) H-CDR1, H- comprising CDR2 and H-CDR3, with H-CDR1 comprising the sequence NYDIS [SEQ ID NO:22] (18E12 H-CDR1); H-CDR2 comprising the sequence VIWTSGGTNYNSAFMS [SEQ ID NO:23] (18E12 H-CDR2); and An antibody comprising an antibody VH domain, or an antigen-binding fragment thereof, wherein H-CDR3 comprises the sequence GDGNFYLYNFDY [SEQ ID NO:24] (18E12 H-CDR3).

In some embodiments, the CD14 antagonist antibody is humanized. In examples of this type, the humanized CD14 antagonist antibody preferably comprises a donor CDR set corresponding to a CD14 antagonist antibody (e.g., one of the CD14 antagonist antibodies described above), and a human acceptor framework. . The human acceptor framework includes: residues flanking the CDRs; residues at glycosylation sites; rare residues; canonical residues; contact residues between the heavy and light chain variable regions. residues in the Vernier zone; and residues in the overlapping region between the Chothia-defined VH CDR1 and the Kabat-defined first heavy chain framework. It may contain at least one amino acid substitution compared to the human germline acceptor framework. Techniques for producing humanized mAbs are well known in the art (eg, Jones et al., 1986. Nature 321: 522-525; Riechmann et al. 1988. Nature 332:323-329; Verhoeyen et al. Carter et al., 1992. Proc.Natl.Acad.Sci.USA 89:4285-4289;Sandhu, JS., 1992.Crit.Rev.Biotech. 12:437 -462, and Singer et al., 1993. J. Immunol. 150: 2844-2857). A chimeric or murine monoclonal antibody can be humanized by transferring the murine CDRs from the heavy and light variable chains of the murine immunoglobulin to the corresponding variable domains of a human antibody. Mouse framework regions (FR) in the chimeric monoclonal antibody are also replaced by human FR sequences. As simple transfer of mouse CDRs to human FRs often results in reduced or even loss of antibody affinity, additional modifications are required to restore the original affinity of mouse antibodies. obtain. This can be accomplished by substituting one or more human residues in the FR region by their murine counterparts to obtain an antibody with good binding affinity for that epitope. For example, Tempest et al. (1991. Biotechnology 9:266-271) and Verhoeyen et al. (1988, supra). In general, human FR amino acid residues that differ from their murine counterparts and that are located close to or contacting one or more CDR amino acid residues are candidates for substitution.

In a preferred embodiment, the CD14 antagonist antibody is the IC14 antibody (Axtelle et al., 2001. J. Endotoxin Res. 7: 310-314; and US Patent Application No. 2006/0121574, incorporated herein by reference in its entirety). is incorporated herein) or an antigen-binding fragment thereof. The IC14 antibody is a chimeric (mouse/human) monoclonal antibody that specifically binds to human CD14. The murine parental antibody for this antibody is 28C5, described above (Leturcq et al. Patent Nos. 5,820,858, 6,444,206 and 7,326,569). and Leturcq et al., 1996. J. Clin. Invest. 98: 1533-1538). The IC14 antibody comprises a VL domain and a VH domain:

The VL domain has the amino acid sequence:
METDTILLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRASNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 25]
includes; and

A VH domain has the amino acid sequence:
MKVLSLLYLLTAIPGILSDVQLQSGPGLVKPSQSLSLTCTVTGYSITSDSSAWNWIRQFPGNRLEWMGYISYSGSTSSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLRFAYWGQGTLVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK [SEQ ID NO: 26]
including.

2.2 Screening Methods The present invention also provides methods for identifying antagonist antibodies of CD14 suitable for use in treating the development or progression of neurodegenerative diseases or symptoms thereof. These methods generally involve determining whether the test substance is capable of directly antagonizing CD14. For example, a method may involve determining whether a test substance is capable of inhibiting or reducing the amount or agonist activity of CD14, wherein the ability to inhibit or reduce the amount or agonist activity of CD14 is It indicates that the substance may be suitable for use in treating the development or progression of a neurodegenerative disease or a symptom thereof as described herein. In some embodiments, the test substance is combined with CD14, or a cell expressing CD14 on its surface, or a nucleic acid sequence in which CD14 is expressed, preferably in the presence of a CD14 agonist such as DAMP or PAMP. A decrease in the amount of CD14 or agonist activity in the presence of agonist when contacted and compared to controls indicates that the test substance binds to and directly antagonizes CD14. Reduction or inhibition of CD14 agonist activity can result in activation of downstream pathways, such as TLR signaling pathways (e.g., TLR4 signaling pathways) and TRIF pathways, or cellular responses (e.g., pro-inflammatory effects such as proinflammatory cytokines). production of sexual mediators). In one embodiment, binding of the antagonist antibody to mCD14-expressing peripheral cells or sCD14 in the subject inhibits or reduces production of one or more pro-inflammatory cytokines associated with disease pathology.

The screening methods of the invention can be performed in vivo, ex vivo or in vitro. In particular, the step of contacting the test substance with CD14 or cells expressing CD14 on their surface (eg, immune cells) can be performed in vivo, ex vivo or in vitro. The screening methods of the invention can be performed in cell-based or cell-free systems. For example, the screening methods of the present invention include the steps of contacting cells expressing CD14 on their surface with a test substance and determining whether contacting the cells with the test substance leads to a decrease in the amount of CD14 or agonist activity. can include

In such cell-based assays, the CD14 and/or test agent may be endogenous to the host cell, introduced into the host cell or tissue, causing or allowing expression of the expression construct or vector. It may be introduced into a host cell or tissue by activating, or by stimulating or activating expression from an endogenous gene in the cell. In such cell-based methods, the amount of CD14 activity is determined whether the agent alters the amount of CD14 in the cell, such as through modulation of CD14 expression in the cell or destabilization of CD14 protein in the cell. , or in the presence or absence of a test substance to determine whether it alters the CD14 agonist activity of the cells. The presence of lower CD14 agonist activity or a decrease in the amount of CD14 on the cell surface in the presence of the test substance indicates that the test substance is for use according to the present invention in treating an individual with a neurodegenerative disease or a symptom thereof. It indicates that it may be a suitable CD14 antagonist.

In one embodiment, such cell-based assays may be performed in vitro or ex vivo on cells or tissues derived from the patient to be treated. Thus, it can be determined whether a test substance can decrease the activity or amount of CD14 in the subject's cells. In one embodiment, the cells are stem cells or macrophages.

In a preferred embodiment, the method is such that the test substance binds to another cellular component, preferably a binding partner of CD14, such as a secreted (e.g. MD2) or cell membrane located (e.g. TLR4) CD14 binding partner. further comprising determining whether the test substance lacks substantial or detectable binding to CD14, thereby determining that the test substance is a specific antagonist of CD14. In a non-limiting example of this type, in the presence of a CD14 agonist such as DAMP or PAMP, a test substance is administered to (1) wild-type cells (e.g., immune cells such as macrophages) that express CD14 on their surface; ), and (2) CD14-negative cells (eg, the same immune cells as in (1) but with loss of function of the CD14 gene). If a test substance inhibits CD14 agonist activity of wild type cells but not CD14 negative cells, this indicates that the test substance is a CD14 specific antagonist. Cells of this type can be constructed using routine procedures or animals.

In other embodiments, the screening methods of the invention may employ cell-free assays. For example, CD14 can be present in a cell-free environment. Suitable cell-free assays can be performed in cell extracts. For example, the contacting step of the methods of the invention may be performed in an extract obtained from cells that may express, produce or otherwise contain CD14 and/or the test substance. A cell-free system comprising CD14 can be incubated with other components of the methods of the invention, such as test substances.

The contacting step of the method of the invention can involve incubation of various components. Such incubations can be performed at any suitable temperature, typically between 4°C and 40°C. Incubation periods may be selected for optimal activity, but may also be optimized to facilitate rapid high-throughput screening. After the contacting and optional incubation steps, the subject method may further include a washing step to remove unbound components, such washing steps generally removing the label giving rise to background signal during detection. For example, it is used where required to remove non-specific binding components that are radioactively or fluorescently labeled.

Incubation in cell-based or cell-free assay systems can be performed in microtiter plates, such as 96-well plates or other microwell plates. Additionally, incubation can be performed in an automated fashion (eg, for high-throughput screening).

The screening methods of the invention can be performed in vivo. For example, screening methods can be performed in animal models. In such in vivo models, the effects of test substances can be assessed in the circulation (eg, blood) or in other organs such as liver, kidney or heart. Preferably the animal is a non-human animal such as a mouse or rat. Such models can be used to assess the in vivo effects of test substances. For example, such models can be used to assess whether a test substance can decrease the activity or amount of CD14 in vivo. In such methods, the amount and/or agonist activity of CD14 can be assessed.

In vivo models can also be used to determine whether a test substance has any undesirable side effects. For example, the methods of the invention use the effects of a test substance on CD14 to determine whether it is specific to other receptors or cellular components (e.g., CD14 binding partners such as MD2 and TLR4). can be compared with its effect on In vivo animal models for MND are well known to those skilled in the art.

In vivo models described herein, or in vitro models such as the cell-based or cell-free assay models described herein, the effect of a test substance on CD14 is determined by CD14 binding, such as MD2 and TLR4. The effect of the same substance on cellular components such as partners can be compared. As discussed above, desirable CD14 antagonist antibodies for use in the therapeutic and prophylactic methods described herein can be antibodies that specifically antagonize CD14. Screening methods of the invention may therefore include the additional step of assessing whether the test substance has any effect on the activity or amount of one or more other such cellular components. In such methods, the test substance decreases the activity or amount of CD14 but does not significantly decrease the activity or amount of one or more other cellular components, such as CD14 binding partners such as MD2 and TLR4. A suitable CD14 antagonist antibody may be identified if it is found not to decrease, not significantly decrease, not alter, or alter the lining.

In the screening methods described herein, the presence of lower CD14 agonist activity or a decrease in the amount of CD14 in the presence of a test agent indicates that the test agent is associated with the development or progression of a neurodegenerative disease or symptoms thereof in an individual. It indicates that there may be suitable antagonist antibodies of CD14 for use in accordance with the present invention for treatment.

A test substance that is an antagonist of CD14 is at least 5%, at least 10%, at least 25%, at least 50%, at least 60%, at least 75% greater in the presence of the test substance than in the absence of the test substance , or may result in a reduction in CD14 activity or levels of at least 85% or greater. A test substance that is an antagonist of CD14 may result in a decrease in CD14 agonist activity or levels such that CD14 agonist activity or levels are no longer detectable in the presence of the test substance. Such a reduction may be seen in the sample being tested or, for example, when the method is performed in an animal model, particularly the circulation or other organs such as the liver, kidney or heart. may be found in tissues from

A test substance that is an antagonist of CD14 is preferably a specific antagonist of CD14 as described above. However, this does not mean that a specific antagonist of CD14 is completely free of off-target antagonist activity. In this regard, specific antagonists of CD14 may have negligible or minimal direct binding and effects on other cellular components, thereby antagonizing the activity, signaling or expression of non-CD14 cellular components. is less than 15%, less than 10%, less than 5%, less than 1%, or less than 0.1% of the direct binding and effect of the substance on CD14 activity, signaling or expression.

The level or amount of CD14 can be measured by assessing the expression of the CD14 gene. Gene expression can be assessed by examining mRNA production or levels or protein production or levels. Expression products such as mRNA and proteins can be identified or quantified by methods known in the art. Such methods may utilize hybridization to specifically identify the mRNA of interest. For example, such methods may involve PCR or real-time PCR approaches. A method of identifying or quantifying a protein of interest may involve the use of an antibody that binds to the protein. For example, such methods may involve Western blotting. Modulation of CD14 gene expression can be compared in the presence and absence of the test substance. Thus, test agents can be identified that decrease CD14 gene expression relative to levels seen in the absence of the test agent. Such test substances may be suitable antagonists of CD14 according to the invention.

Screening methods can assess agonist activity of CD14. For example, such methods can be performed using peripheral blood mononuclear cells. Such cells produce cytokines such as IL-1α, IL-6, TNF-α, IFN-β, IL-1β, IL-17 and IL-8 in response to stimulation with, for example, LPS. . Screening methods may therefore include combining peripheral blood mononuclear cells with a test substance or carrier and adding LPS. Cells can then be incubated for a length of time (eg, 24 hours) to allow production of pro-inflammatory mediators such as cytokines. Levels of cytokines such as IL-1α, IL-6, TNF-α, IFN-β, IL-1β, IL-17 and IL-8 produced by the cells during that period can then be assessed. If the test substance has anti-CD14 properties, the production of such cytokines should be reduced compared to carrier-treated cells.

Further tests may also be performed to confirm that the test substance is suitable for use in the claimed methods. For example, as noted above, suitable antagonist antibodies of CD14 should be able to reduce the detrimental consequences of pro-inflammatory mediator production (also commonly referred to as cytokine storm). The screening methods of the invention are therefore useful for assessing the effect of a test substance in animals with such production of pro-inflammatory mediators (eg, animals with ALS) and viewing that effect in the absence of the test substance. Further steps such as those discussed above may be incorporated, involving comparing to the effect obtained. Suitable CD14 antagonists can ameliorate at least part of the effects of neurodegenerative diseases in test animals.

2.3 Adjunctive anti-neurodegenerative agents As indicated, the compounds according to the invention may be used alone or with other agents (sometimes referred to herein as "adjunctive anti-neurodegenerative agents"). Other agents that may be administered in combination, particularly other compounds of the invention or direct CD14 antagonists that are not and otherwise useful for the treatment of the onset or progression of neurodegenerative diseases or symptoms thereof. Compounds disclosed as being are included. For MNDs such as amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, primary lateral sclerosis, progressive muscular atrophy, pseudobulbar palsy, progressive bulbar palsy and lower motor neuron disease , non-limiting examples of adjunctive anti-neurodegenerative agents contemplated herein include riluzole (disclosed in Miller RG et al. The Cochrane Database of Systematic Reviews. 3: CD001447); agents that block interaction with CD40 ligand, such as antibodies that specifically bind to CD40 and/or CD40 ligand (eg, AT-1502), and anti-inflammatory agents, such as complement pathway blockers, such as C5a receptor agonists such as PMX205 or eculizumab (JD et al, (2017) British Journal of Pharmacology, 174(8)). Examples include NurOwn Stem Cell Therapy (BrainStorm Cell Therapeutics), GM604, Radicava (Radicat), Macitinib, Memantine or Tiracemtib Examples contemplated herein for dementias such as LBD, FTD or AD Additional adjunctive anti-neurodegenerative agents are cholinesterase inhibitors such as donepezil (Aricept), rivastigmine (Exelon) and galantamine (reminyl and lazadaine). Agents include, but are not limited to, approved therapeutic agents selected from, for example, Aricept, Lazadaine, Namenda, Exelon, and Namzaric, for use in combination therapy with the CD14 antagonist antibodies described herein. The selection of a suitable adjunctive anti-neurodegenerative agent for is well within the skill of the art.

Supplementary anti-neurodegenerative agents may be used for their additive activity or therapeutic profile in neurodegenerative diseases (e.g., MND and dementia) and, in certain instances, for synergistic effects in combination with the compounds of the invention. For this purpose, it can be used in combination with a CD14 antagonist antibody.

If combination therapy is desired, the CD14 antagonist antibody is administered separately, simultaneously or sequentially with the adjuvant. In some embodiments, this is achieved by systemically administering a single composition or pharmacological formulation comprising both types of formulations, or one composition comprising a CD14 antagonist antibody and the other This can be accomplished by administering two separate compositions or formulations containing the adjuvant simultaneously. In other embodiments, treatment with the CD14 antagonist antibody may precede or follow treatment with the adjuvant by intervals ranging from minutes to days.

In embodiments in which the CD14 antagonist antibody is applied separately from the adjuvant, generally no long period of time elapses between each delivery point so that the CD14 antagonist antibody is still administered peripherally with the adjuvant. Combined effect on inhibition of CD14-mediated effects, such as inhibition or reduction of pro-inflammatory mediator production by cells (e.g., immune cells such as, but not limited to, macrophages, monocytes, dendritic cells or T cells) In particular, it ensures that a combined effect can be advantageously exerted to maintain or enhance the subject's ability to reverse or inhibit the onset of the disease or its symptoms.

In some situations, both modalities can be administered within about 1-12 hours of each other, more preferably within about 2-6 hours of each other. In some circumstances, it may be desirable to extend the period for treatment significantly, however several hours (2, 3, 4, 5, 6 or 7) to several days (1, 3, 4, 5, 6 or 7) between each administration. 2, 3, 4, 5, 6, 7 or 8) elapses. In certain embodiments, the adjuvant is administered prior to administration of the CD14 antagonist antibody.

In embodiments in which the adjuvant is administered separately from the CD14 antagonist antibody, depending on the neurodegenerative agent and the disease or symptom thereof being treated, the adjuvant will be administered in a different manner than that used for the CD14 antagonist antibody. For example, it will be understood that the adjuvant may be administered systemically or directly to the CNS.

It can be assumed that more than one administration of either the CD14 antagonist antibody or adjuvant is desired. Various combinations can be used with the CD14 antagonist antibody as "A" and the adjuvant as "B", as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/ B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B.

When two or more therapeutic agents are administered "in conjunction" or "concurrently" to a subject, they may be administered simultaneously in a single composition, or simultaneously in separate compositions, or separately. can be administered at separate times in the composition of

The subject methods can also be supplemented with or practiced in combination with other medical interventions such as via tablets, oral solutions, patches or intravenous injections or other parenteral modes of administration. For example, in neurodegenerative diseases that lead to decreased lung function, such as ALS, intervention may be mechanical, such as non-invasive ventilation devices, or drugs are used to relieve dyspnea. good. The subject methods can also be practiced in combination with non-medical therapies including, but not limited to, physical therapy, speech-language therapy, psychotherapy, occupational therapy.

2.4 Compositions As described herein, the use of CD14 antagonist antibodies, either alone or in combination with adjunctive anti-neurodegenerative agents, is effective in binding to cells expressing mCD14. or through binding to sCD14 can inhibit or reduce the production of pro-inflammatory mediators such as pro-inflammatory cytokines from peripheral cells and can reduce the sequelae of that production, more specifically In particular, the development or progression of neurodegenerative diseases and their symptoms can be treated.

The CD14 antagonist antibody and optionally ancillary anti-neurodegenerative agents can be administered alone or with a pharmaceutically acceptable carrier.

For use in the methods described herein, the CD14 antagonist antibody may be administered in a conventional manner using one or more pharmaceutically acceptable carriers, stabilizers or excipients (carriers). may be formulated to form pharmaceutical compositions as is known in the art, particularly for protein active agents. A carrier is "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient (eg, the patient) thereof. Suitable carriers typically include saline or polyols such as ethanol glycerol or propylene glycol.

Antibodies can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups), which are formed with inorganic acids such as hydrochloric or phosphoric acid, or with acetic, oxalic, tartaric and maleic acids. formed with organic acids such as Salts formed with free carboxyl groups also include inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine and procaine. It can be derived from bases.

The compositions may suitably be formulated for systemic administration, such as intravenous, intramuscular, subcutaneous, or intraperitoneal administration, conveniently containing a sterile antibody, preferably isotonic with the blood of the recipient. contains an aqueous solution of Such formulations typically contain physiologically compatible substances such as sodium chloride, glycine, and the like, and solid active agents in water having a buffered pH compatible with physiological conditions. It is prepared by dissolving the ingredients to produce an aqueous solution and sterilizing said solution. They can be prepared in unit or multi-dose containers, such as sealed ampoules or vials.

The composition may incorporate stabilizers such as, for example, polyethylene glycols, proteins, saccharides (eg, trehalose), amino acids, inorganic acids and mixtures thereof. Stabilizers are used in aqueous solutions of appropriate concentration and pH. The pH of the aqueous solution is adjusted within the range of 5.0-9.0, preferably within the range of 6-8. Antiadsorbents may be used in the formulation of antibodies. Other suitable excipients may typically include antioxidants such as ascorbic acid. The composition may be formulated as a controlled release preparation, which may be accomplished through the use of protein-conjugated or absorbing polymers. Suitable polymers for controlled release formulations include, for example, polyesters, polyamino acids, polyvinyls, pyrrolidone, ethylene vinyl acetate, and methylcellulose. Another possible method for controlled release is to incorporate the antibody into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinyl acetate copolymers. Alternatively, instead of incorporating these agents into polymer particles, e.g., in microcapsules prepared by coacervation techniques or interfacial polymerization, e.g. It is possible to entrap these materials in microcapsules, or colloidal drug delivery systems, such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, or macroemulsions.

A CD14 antagonist antibody and optionally an adjunctive anti-neurodegenerative agent can also be administered directly to the respiratory tract in the form of an aerosol. For use as an aerosol, the inhibitor of the invention in solution or suspension can be combined with a suitable propellant, for example a hydrocarbon propellant such as propane, butane, or isobutane, together with a conventional adjuvant into a pressurized aerosol. It can be packaged in a container. The materials of the invention can also be administered in non-pressurized form, such as in a nebulizer or atomizer.

Those skilled in the art will recognize that formulations are routinely designed according to their intended use, ie route of administration.

3. Methods of Treatment The present invention provides methods of treatment for treating a subject having or at risk of developing (or susceptible to) a neurodegenerative disease. These methods include within their scope the development or progression of neurodegenerative diseases in humans and animals (eg, veterinary applications), as well as treatment of symptoms associated with such diseases. Such diseases include, for example, MNDs selected from ALS, PLS, PMA, progressive bulbar palsy (PBP) and pseudobulbar palsy. Such diseases also include, for example, dementias selected from Alzheimer's disease, dementia with Lewy bodies (DLB and PDD), frontotemporal dementia (FTD) and vascular dementia.

The present invention contemplates a method of treating the development or progression of a neurodegenerative disease or a symptom thereof in a subject by systemically administering to the subject a CD14 antagonist antibody of the present invention and, optionally, an adjunctive anti-neurodegenerative agent. . A CD14 antagonist antibody, and optionally an adjunctive anti-neurodegenerative agent (sometimes referred to herein as a "therapeutic agent") may be used for its intended purpose, such as alleviation of symptoms associated with the disease. can be administered in an "effective amount" to achieve a The dose of therapeutic agent administered to a patient should be sufficient to achieve a beneficial response in the subject over time, such as a reduction in at least one symptom associated with a neurodegenerative disease.

In one embodiment, the disease is MND and progressive muscle wasting, paralysis, spasticity, hyperreflexia, respiratory and swallowing difficulties, leg weakness, dysarthria, slurred speech, gait disturbance, facial weakness and at least one symptom selected from other symptoms such as muscle cramps are reduced. In one embodiment, the disease is dementia and memory loss; depression, communication disorders, poor judgment, disorientation, confusion, behavioral changes, motor symptoms, hallucinations, neuroleptic hypersensitivity and speech, swallowing and At least one symptom selected from difficulty walking is reduced.

The amount of therapeutic administered or frequency of administration may depend on the subject being treated, including the age, sex, weight and general health of the subject. In this regard, the precise amount of therapeutic agent to administer depends on the judgment of the physician. Through routine experimentation, one skilled in the art will know which CD14 antagonist antibodies described herein and, optionally, adjunctive anti-neurodegenerative antibodies to be included in the pharmaceutical compositions of the invention for the desired therapeutic outcome. An effective, non-toxic amount of the agent can be determined.

In some embodiments, an "effective amount" of a therapeutic agent is an amount that contains the active ingredient in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount inhibits or reduces the production of one or more pro-inflammatory mediators to treat symptoms of neurodegenerative disease (e.g., ALS or AD) and is treated means the amount of active ingredient (CD14 antagonist) effective to prolong the survival of the subject in question. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

Subjects having or at risk for a neurodegenerative disease include patients who have one or more biomarkers of disease severity or are indicative of a susceptibility to developing a neurodegenerative disease. In one embodiment, the disease is MND and the biomarkers are, for example, SOD1, TDP-43, FUS, C9ORF72, ALS2, ALS4, ALS8, NEK1, UBQLN2, VCP, SETX, ANG, PFN1, MATR3, CHCHD10 , TUBA4A, TBK1, C21orf2 and OPTN or one or more of their expression products. In this embodiment, the presence of markers of neurodegenerative disease is preferably the presence or overexpression of marker gene expression products and/or marker genes (e.g. SOD1, TDP-43, FUS, C9ORF72, ALS2) in the biological sample. , ALS4, ALS8, NEK1, UBQLN2, VCP, SETX, ANG, PFN1, MATR3, CHCHD10, TUBA4A, TBK1, C21orf2 and OPTN mRNA or polypeptide). In some embodiments for MND, the presence of cytoplasmic deposits of TDP-43 positive inclusions and/or elevated serum and/or CSF levels of neurofilaments can also be determined.

In another embodiment, the disease is dementia such as Alzheimer's disease, FTD LBD (DLB and PDD) and vascular dementia, and the marker encodes amyloid precursor protein (APP) and presenilins 1 and 2 mutations in the gene, mutations in the ε4, 2 and 3 alleles of the apolipoprotein E (APOE) gene (APOE-ε4, APOE-ε2, APOE-ε3), Triggering receptor expressed on myeloid cells 2 (TREM2) gene, MAPT The gene is selected from one or more of mutations in the gene, the GRN gene, also referred to as the PGRN gene, the TARDBP gene, the VCP gene and the CHMP2B gene. Elevated serum and/or CSF levels of α-synuclein, S100A9 and S100B, chromogranin, circulating DNA, heat shock proteins and amyloid can also be determined.

In other embodiments, a subject at risk for neurodegenerative disease also has pro-inflammatory cytokines associated with disease in peripheral blood mononuclear cells (PBMCs), such as TNF, IL-1-α, IL-6, one or more of IFN-β, IL-1β, IL-8, IL-18, C-reactive protein (CRP), IL-17, chemokines, CD14-high monocytes and inflammatory mediator mRNA transcripts Identification can be made by determining the presence of elevated levels. In one embodiment, the disease is MND and the cytokine is selected from one or more of IL-6 or IL-17. In one embodiment the disease is dementia and the cytokine is selected from IL-1, IL-6 and TNF-α.

It will be appreciated that any known biomarker of neurodegenerative disease can be used to identify a subject having or at risk of disease.

Administration of the prophylactic agent can occur prior to the onset of symptoms characteristic of a neurodegenerative disease, thereby inhibiting the disease or alternatively slowing its progression.

In order that the present invention may be easily understood and effected in practice, certain preferred embodiments will now be described by way of the following non-limiting examples.

Example 1
Treatment and Prevention of Patients with MND by Systemic Administration of Monoclonal Antibody IC14 Study Design Familial or sporadic MND (clinically likely, probable or definite ), presenting first symptoms of MND within 3 years of informed consent, and aged between 18 and 75 years will be recruited.

Dosing Regimens Patients were blindly assigned to receive 1 of 2 dose regimens of IC14, with all doses delivered IV as an infusion over a 2 hour period:
• IC14 at a dose of 2 mg/kg on study day 1, followed by 1 mg/kg QD on study days 3-5 for a total of 4 doses of IC14 for the first 3 patients.
• IC14 at a dose of 4 mg/kg on study day 1, followed by 2 mg/kg QD on study days 2-4 for a total of 4 doses of IC14 for 7 subsequent patients.

Participation in the study continues for 28 days after the last dose of study drug.

Dosage form:
Sterile glass vials containing IC14 at 5 mg/mL in 30 mL vials (125 mg in 25 mL delivered). The test drug is prepared in 250 mL of sterile normal saline for injection that is infused intravenously over 2 hours.

Study endpoints:
The primary endpoints of the study are the safety, tolerability and lack of immunogenicity of IC14. The safety and tolerability of IC14 are determined by examining toxicities and adverse events attributable to treatment. Safety parameters include evaluation of clinical signs and symptoms from history and physical examination, vital signs, adverse events, and laboratory findings (Chem-20, CBC, platelet count, coagulation test). Immunogenicity studies measure antibodies against IC14.

Secondary endpoints are:
- Peak serum IC14 concentration after administration of starting dose, peak serum and cerebrospinal fluid (CSF) concentrations after course of treatment and area under the serum IC14 concentration versus time curve (AUC) after administration of starting dose and course of treatment .
Relevant to treatment in disease severity by the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R), forced vital capacity (FVC) in the sitting position and other RFT parameters and respiratory function by the Nasal Inspiratory Pressure (SNP) test change.
• Treatment-related changes in quality of life by the ALS Specific Quality of Life-Revised (ALSSQOL-R) score and cognitive function by the Edinburgh Cognitive and Behavioral Assessment (ECAS) score.
• Treatment-related changes in disease biomarker profiles including CRP, IL-6, IL-17, IL-1β and neurofilament heavy and light chains.

Specimen Collection and Patient Evaluation:
Samples are collected from patients as detailed in Table 2:

Data Analysis MND biomarkers such as transactivation response element (TAR)-DNA binding protein (TDP)-43 and neurofilament plasma concentrations are measured. Serum and CSF neurofilaments may be assessed.

Other potential biomarkers one or more of which may be assessed include CRP, SOD1, IL-1β, IL-6, IL-17, chemokines, CD14-high monocytes and peripheral blood mononuclear cells (PBMC) inflammatory mediator mRNA transcripts. Serum and CSF are evaluated in vitro for their ability to inhibit inflammatory gene activation.

Pharmacodynamic evaluation is performed by measuring saturation of CD14 on monocytes and levels of circulating sCD14 at baseline and immediately following doses 1 and 4 of study drug. Pharmacokinetic measurements of IC14 in serum are taken at baseline; immediately and 6 and 22 hours after dose 1; before and immediately and 24 hours after dose 4; and at washout. Pharmacokinetic measurements of IC14 in CSF are performed at baseline, after the fourth dose of IC14 and during washout.

Participation in the study continues for 28 days after the last dose of study drug. Study participation will be for a total of 32-33 days plus a maximum of 4 weeks for screening evaluations.

Study days are defined as consecutive calendar days from the start of the first study drug administration (Study Day 1). At approximately 24 hour intervals (or 48 hour intervals between the first and second doses for the first 3 patients receiving the first dose) from the start of the first study drug administration (Study Day 1) Administer study drug.

Information was collected on the pharmacodynamics and pharmacokinetics of IC14 in the blood and CSF of MND patients, including:
• Baseline, peak and washout serum IC14 concentrations after administration of the starting dose, and baseline, peak and washout serum and cerebrospinal fluid (CSF) concentrations after the course of treatment.
• Area under the serum IC14 concentration vs. time curve (AUC) after administration of the starting dose and after the course of treatment.
• Circulating monocyte CD14 receptor binding and sCD14±sCD14-ST (preseptin) levels are calculated at baseline and after Dose 1 and Dose 4.

Information on treatment-related changes in disease biomarker profiles in peripheral blood and CSF, such as levels of immune cells, inflammatory cytokines and chemokines, and changes in other biomarkers such as neurofilament (NF) heavy and light chain levels .

Compounds Test drug IC14 was obtained from Implicit Bioscience Ltd. (Queensland, Australia). IC14 is a recombinant chimeric (mouse/human) monoclonal antibody against human CD14. The mouse parental antibody is the antibody designated 28C5. IC14 is secreted from Chinese hamster ovary cells as an L2H2γ4 immunoglobulin.

Results Results for a single patient given a total dose of IC14, i.e. 4 mg/kg on study day 1 followed by 2 mg/kg QD on study days 2-4 for a total of 4 doses got Lumbar punctures were performed and CSF collected at baseline and on study days 4 and 8 as described above. IL-17 and IL-1β levels were measured by multiplex analysis, and neurofilament light chain (NFL) levels were measured by ELISA. As shown in FIG. 1, treatment with IC14 resulted in decreased levels of the inflammatory markers IL-17 and IL-1β in CSF on day 8, and also decreased NFL, a marker of disease. rice field. IL-17 and IL-1β have been associated with disease in both ALS/MND patients and ALS/MND disease models (Rentzos et al. (2010) Acta Neurol. Scand. 122, 425-9; Fiala et al. (2010) Neuroinflammation 7, 76; Meissner et al.(2010) Proc.Natl.Acad.Sci.107, 13046-13050;van der Meer & Simon. d.Sci.U. SA 107, 12741-2; and Zhao et al. (2015) Exp. Neurol. 273, 24-35), NFL has been described as a marker of neurodegeneration. The ability of IC14 to modulate the levels of these molecules in the CSF when delivered systemically to subjects with MND suggests that intravenous IC14 therapy may have therapeutic utility in suppressing the neuroinflammatory processes that drive ALS/MND. indicates that it has

Example 2
IC14 Inhibition of TDP-43 Driven Activation of Primary Human Microglial Cells The immune response in the brain and spinal cord is primarily a neuroinflammatory response that acts as a first line of defense and drives neurodegenerative diseases such as ALS. It is mediated by microglia, which play a key role in the initiation and maintenance of cytotoxicity (Salter & Stevens (2017) Nat. Med. 23, 1018-1027).

SOD1 and TDP-43 are intracellular proteins involved in ALS. An in vitro system using mouse microglia showed that both proteins act as damage-associated molecular patterns (DAMPs) to activate microglia, induce pro-inflammatory cytokines, and induce neurotoxicity. It has been proven to promote These responses utilize TLR and NLRP3 signaling and DAMP-driven activation is inhibited by blocking antibodies to TLR2, TLR4 or CD14 (Zhao et al. (2010) Glia 58, 231-243; and Zhao et al. (2015) Exp. Neurol. 273, 24-35), pointing to a role for blockade of CD14 in inhibiting neuroinflammation.

Using a tissue culture system for proliferation of primary human microglia, we determined whether DAMP TDP-43 activates human microglia, thereby providing an inflammatory readout of this activation process. The ability of IC14 to identify and inhibit this activation was assessed.

Materials and Methods Reagents IC14 was provided by Implicit Bioscience Ltd (IC14-3, Lot 1-FIN-0779) and the activity and stability of this GMP antibody was confirmed by stability studies. As a control for IC14, an isotype control human IgG4 antibody was obtained from Australian Biosearch (Ultra-LEAF™ Purified Human IgG4 Isotype, catalog #403402).

Both wild-type and mutant (Q331K) forms of E. Recombinant TDP-43 produced in E. coli was provided by Associate Professor Julie Atkins, Macquarie University.

Culture of Microglia Cultures of primary human microglia were prepared as described by Guillemin et al. (J. Neurosci. Res. (1997) 49, 576-591). Briefly, fetal human brain tissue was collected from 14- to 19-week-old aborted fetuses recovered after therapeutic abortion after obtaining informed consent (approved by the Human Research Ethics Committee of Macquarie University; HREC 5201600719 ). One gram of this material was aliquoted into 24 wells of a 24-well tissue culture plate (Corning) by surgical dissection and incubated at 37°C with 5% Cultured for 5 days in _CO2 . After a 2 minute trypsinization step, non-microglial cells were removed, leaving microglia attached to the plate, by neutralization with DMEM+FCS. Microglia were cultured for an additional 48 hours prior to treatment.

Stimulation of Microglia One-week-old primary microglia cultures were pretreated for 2 hours with either IC14 or control IgG4 mAb antibody at the following concentrations: untreated; 1 μg/ml, 100 ng/ml or 10 ng. IC14 at 1 μg/ml final concentration; control IgG4 at 1 μg/ml, 100 ng/ml, or 10 ng/ml final concentration.

Wild type or mutant TDP-43 was then added to a final concentration of 500 ng/ml. Control wells received no TDP-43.

Cultures were incubated in DMEM containing 10% heat-inactivated FCS at 37° C., 5% CO ₂ for 48 hours. Supernatants were harvested for multiplex cytokine analysis, using a 65-plex cytokine and chemokine array (Human Cytokine/Chemokine 65-Plex Panel, Eve Technologies, Calgary, Alberta), and 3 cells for profiling of kynurenine pathway metabolites. analyzed in series.

Results Cytokine/Chemokine Induction In the absence of any external stimuli, primary human microglia produce a wide range of cytokines and chemokines, including many that have been identified in the circulation or CSF of patients with ALS. (Fig. 2). Most of these molecules were expressed at low levels, but have been shown to be mutated in patients with both familial and sporadic MND and have pro-inflammatory properties (Zhao et al. (2015) Exp. Neurol. . Zhao et al. Minimal differences were detected in the ability of wild-type or mutant TDP-43 to activate microglia, consistent with the observations of . The cytokines and chemokines produced include the proinflammatory cytokines TNFα, IL1α and IL-12p40, as well as the chemokines CXCL10 and CCL5, which play a role in promoting the attraction of inflammatory cells to the site of inflammation (Fig. 2A). and B). Thus, TDP-43 may initiate and sustain motor neuron injury in ALS by activation of microglia, localized production of inflammatory mediators and subsequent attraction of inflammatory cells to the site of neuronal injury. .

Pretreatment of microglial cultures with an isotype control antibody had no effect on this TDP-43 driven cytokine production. Inclusion of IC14 resulted in dose-dependent inhibition of both wild-type and mutant TDP-43-driven cytokine and chemokine production, including inhibition of TNFα, IL-1α, IL12p40 and CXCL10. (Figs. 2A and B). Expression levels following TDP-43 stimulation for many of these analytes were elevated beyond the range of the standard curve (eg TNFα, CXCL10), precluding accurate characterization of expression levels. Nonetheless, inhibition of cytokine or chemokine production was detected under these extreme conditions, suggesting that IC14 may be effective in in vitro assays that utilize more physiological DAMP-driven stimulatory conditions. bottom.

The disclosures of all patents, patent applications, and publications referenced herein are hereby incorporated by reference in their entireties.

Reference to any reference herein shall not be construed as an admission that such reference is available as "prior art" to the present application.

The purpose throughout this specification has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those skilled in the art will therefore appreciate, in light of the present disclosure, that various modifications and changes may be made in the particular embodiments illustrated without departing from the scope of the invention. All such modifications and variations are intended to be included within the scope of the appended claims.

Claims (56)

A method of inhibiting or reducing production of pro-inflammatory mediators from mammalian cells in the periphery of a subject with a neurodegenerative disease, the method comprising removing membrane-bound CD14 (mCD14) or soluble CD14 (sCD14) from the peripheral cells. A method comprising, consisting of or consisting essentially of contacting with a sufficient amount of a CD14 antagonist antibody to inhibit or reduce production of a pro-inflammatory mediator. A method of treating the development or progression of a neurodegenerative disease or a symptom thereof in a subject, the method comprising, consisting of, or consisting essentially of systemically administering to said subject an effective amount of a CD14 antagonist antibody. Use of a CD14 antagonist antibody to treat the development or progression of a neurodegenerative disease, wherein said CD14 antagonist antibody is administered systemically. 4. Use according to claim 3, wherein said CD14 antagonist antibody is manufactured as a medicament for said indication. 5. The method or use of any one of claims 2-4, wherein said CD14 antagonist antibody inhibits or reduces production of one or more pro-inflammatory mediators by peripheral cells in said subject. 6. The method or use of claim 5, wherein said CD14 antagonist antibody binds to both mCD14 and sCD14. 7. The method or use of claim 1, claim 5 or claim 6, wherein said peripheral cells are immune cells. 8. The method or use of claim 7, wherein said immune cells are selected from monocytes, macrophages, dendritic cells or T cells. said proinflammatory mediator is tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1)-α, IL-6, IFN-γ, IFN-β, IL-1β, IL-8, 9. The method or use of any one of claims 1-8, wherein the cytokine is selected from one or more of IL-17 and IL-18. 10. Any one of claims 1 to 9, further comprising identifying that said subject has or is at risk of developing a neurodegenerative disease, preferably prior to administering said CD14 antagonist antibody. any method or use of any of the preceding paragraphs. A method or use according to any preceding claim, wherein said neurodegenerative disease or disorder is selected from MND or dementia. The MND is selected from the group consisting of amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), and pseudobulbar palsy. 12. A method or use according to claim 11, wherein 13. A method or use according to claim 12, wherein said MND is ALS. 12. The method or use of claim 11, wherein said dementia is selected from the group consisting of Alzheimer's disease, dementia with Lewy bodies, frontotemporal dementia (FTD) and vascular dementia. The CD14 antagonist antibody is
(1) Array:
QSPASLAVSLGQRATISCRASESVDSFGNSFMHWYQQKAGQPPKSSIYRAANLESGIPARFSGSGSRTDFTLTINPVEADDVATYFCQQSYEDPWTFGGGTKLGNQ [SEQ ID NO: 1]
a VL domain (3C10 VL) comprising, consisting of or consisting essentially of; and the sequence:
LVKPGGSLKLSCVASGFTFSSYAMSWVRQTPEKRLEWVASISSGGTTYYPDNVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARGYYDYHYWGQGTTLTVSS [SEQ ID NO: 2]
a VH domain (3C10 VH) comprising, consisting of or consisting essentially of
an antibody comprising;
(2) Array:
QSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRASNLQS GIPARFSGSGSRTDFTLTINPVEADDVATYCCQQSNEDPTTFGGGTKLEIK [SEQ ID NO: 3]
a VL domain (28C5 VL) comprising, consisting of or consisting essentially of; and the sequence:
LQQSGPGLVKPSQSLSLTCTVTGYSITSDSAWNWIRQFPGNRLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLRFAYWGQGTLVTVSA [SEQ ID NO: 4]
a VH domain comprising, consisting of or consisting essentially of (28C5 VH)
and (3) the sequence:
QTPSSLSASLGDRVTISCRASQDIKNYLNWYQQPGGTVKVLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQRGDTLPWTFGGGTKLEIK [SEQ ID NO: 5]
a VL domain (18E12 VL) comprising, consisting of or consisting essentially of; and the sequence:
LESGPGLVAPSQSLSITCTVSGFSLTNYDISWIRQPPGKGLEWLGVIWTSGGTNYNSAFMSRLSITKDNSESQVFLKMNGLQTDDTGIYYCVRGDGNFYLYNFDYWGQGTTLTVSS [SEQ ID NO: 6]
a VH domain (18E12 VH) comprising, consisting of or consisting essentially of
15. A method or use according to any one of claims 1 to 14, selected from antibodies, including 16. A method or use according to claim 15, wherein said CD14 antagonist antibody comprises, consists of or consists essentially of the VL and VH CDR sequences of said antibody as defined in claim 13. The CD14 antagonist antibody is
(1) a) comprising L-CDR1, L-CDR2 and L-CDR3, where L-CDR1 comprises the sequence RASESVDSFGNSFMH [SEQ ID NO: 7] (3C10 L-CDR1); L-CDR2 comprises the sequence RAANLES [SEQ ID NO: 8] (3C10 L-CDR2); and L-CDR3 comprises the sequence QQSYEDPWT [SEQ ID NO:9] (3C10 L-CDR3); and b) H-CDR1, H- comprising CDR2 and H-CDR3, with H-CDR1 comprising the sequence SYAMS [SEQ ID NO: 10] (3C10 H-CDR1); H-CDR2 comprising the sequence SISSGGTTYYPDNVKG [SEQ ID NO: 11] (3C10 H-CDR2); and an antibody comprising an antibody VH domain, or an antigen-binding fragment thereof, wherein H-CDR3 comprises the sequence GYYDYHY [SEQ ID NO: 12] (3C10 H-CDR3);
(2) a) comprising L-CDR1, L-CDR2 and L-CDR3, where L-CDR1 comprises the sequence RASESVDSYVNSFLH [SEQ ID NO:13] (28C5 L-CDR1); L-CDR2 comprises the sequence RASNLQS [SEQ ID NO:14] (28C5 L-CDR2); and L-CDR3 comprises the sequence QQSNEDPTT [SEQ ID NO: 15] (28C5 L-CDR3); and b) H-CDR1, H- comprising CDR2 and H-CDR3, with H-CDR1 comprising the sequence SDSAWN [SEQ ID NO: 16] (28C5 H-CDR1); H-CDR2 comprising the sequence YISYSGSTSYNPSLKS [SEQ ID NO: 17] (28C5 H-CDR2); and an antibody comprising an antibody VH domain, or an antigen-binding fragment thereof, wherein H-CDR3 comprises the sequence GLRFAY [SEQ ID NO: 18] (28C5 H-CDR3); and (3) a) L-CDR1, L-CDR2 and L- L-CDR1 comprises the sequence RASQDIKNYLN [SEQ ID NO:19] (18E12 L-CDR1); L-CDR2 comprises the sequence YTSRLHS [SEQ ID NO:20] (18E12 L-CDR2); an antibody VL domain, or antigen-binding fragment thereof, comprising the sequence QRGDTLPWT [SEQ ID NO: 21] (18E12 L-CDR3); and b) comprising H-CDR1, H-CDR2 and H-CDR3, wherein H-CDR1 comprises the sequence NYDIS [ SEQ ID NO:22] (18E12 H-CDR1); H-CDR2 contains the sequence VIWTSGGTNYNSAFMS [SEQ ID NO:23] (18E12 H-CDR2); and H-CDR3 contains the sequence GDGNFYLYNFDY [SEQ ID NO:24] (18E12 H- 17. A method or use according to claim 16, selected from antibodies comprising an antibody VH domain comprising CDR3), or an antigen-binding fragment thereof. 18. The method or use of claim 17, wherein said CD14 antagonist antibody is humanized. said CD14 antagonist antibody comprises a VL domain and a VH domain,
The VL domain has the amino acid sequence:
METDTILLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRASNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 25]
and said VH domain has the amino acid sequence:
MKVLSLLYLLTAIPGILSDVQLQSGPGLVKPSQSLSLTCTVTGYSITSDSSAWNWIRQFPGNRLEWMGYISYSGSTSSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLRFAYWGQGTLVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK [SEQ ID NO: 26]
including,
19. A method or use according to claim 18. 20. The method or use of claim 19, wherein said CD14 antagonist antibody is IC14, or an antigen-binding fragment thereof. 21. The method of any one of claims 1-20, wherein said CD14 antagonist is administered in combination with one or more adjunctive anti-neurodegenerative agents to treat said neurodegenerative disease or said symptom thereof, or use. 22. The method or use of claim 21, wherein said CD14 antagonist antibody and said supplemental anti-neurodegenerative agent are administered in synergistically effective amounts. 23. The method or use of claim 22, wherein said CD14 antagonist antibody and said supplemental anti-neurodegenerative agent are administered simultaneously or sequentially. 24. A method or use according to claim 23, wherein said adjunctive anti-neurodegenerative agent is an anti-inflammatory agent. 25. The method or use of claim 24, wherein said anti-inflammatory agent is a complement pathway inhibitor. 26. A method or use according to claim 25, wherein said complement pathway inhibitor is a C5a inhibitor. 27. The method or use of claim 26, wherein said C5a inhibitor is PMX205 or eculizumab. 24. A method or use according to claim 23, wherein said supplementary anti-neurodegenerative agent is an agent that blocks the interaction of CD40 with CD40 ligand. 29. A method or use according to claim 28, wherein said agent is an antibody that specifically binds CD40 and/or CD40 ligand. 30. A method or use according to claim 29, wherein said antibody is AT-1502. 21. Use according to claim 20, wherein said adjunctive anti-neurodegenerative agent is riluzole or edaravone. 1. A pharmaceutical composition for treating the progression or development of a neurodegenerative disease, or a symptom thereof, comprising, consisting of, or consisting essentially of a CD14 antagonist antibody together with a pharmaceutically acceptable carrier or diluent, said A composition, wherein the composition is formulated for systemic administration. 33. The pharmaceutical composition of claim 32, wherein said CD14 antagonist antibody binds both sCD14 and mCD14 and inhibits or reduces production of one or more proinflammatory mediators by peripheral cells in the subject. 34. The pharmaceutical composition of claim 33, wherein said cells are immune cells. 35. The pharmaceutical composition according to claim 34, wherein said immune cells are selected from monocytes, macrophages, dendritic cells or T cells. said proinflammatory mediator is tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1)-α, IL-6, IFN-γ, IFN-β, IL-1β, IL-8, 36. The pharmaceutical composition according to any one of claims 32-35, which is a cytokine selected from one or more of IL-17 and IL-18. 37. Any one of claims 32-36, further comprising identifying that the subject has or is at risk of developing a neurodegenerative disease, preferably prior to administering said CD14 antagonist antibody. The pharmaceutical composition according to . The pharmaceutical composition according to any one of claims 32-37, wherein said neurodegenerative disease or disorder is selected from MND or dementia. The MND is selected from the group consisting of amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), and pseudobulbar palsy. 39. The pharmaceutical composition of claim 38, wherein 39. The pharmaceutical composition of claim 38, wherein said MND is ALS. 39. The pharmaceutical composition of claim 38, wherein said dementia is selected from the group consisting of Alzheimer's disease, dementia with Lewy bodies, frontotemporal dementia (FTD) and vascular dementia. The CD14 antagonist antibody is
(1) Array:
QSPASLAVSLGQRATISCRASESVDSFGNSFMHWYQQKAGQPPKSSIYRAANLESGIPARFSGSGSRTDFTLTINPVEADDVATYFCQQSYEDPWTFGGGTKLGNQ [SEQ ID NO: 1]
a VL domain (3C10 VL) comprising, consisting of or consisting essentially of; and the sequence:
LVKPGGSLKLSCVASGFTFSSYAMSWVRQTPEKRLEWVASISSGGTTYYPDNVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARGYYDYHYWGQGTTLTVSS [SEQ ID NO: 2]
a VH domain (3C10 VH) comprising, consisting of or consisting essentially of
an antibody comprising;
(2) Array:
QSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRASNLQS GIPARFSGSGSRTDFTLTINPVEADDVATYCCQQSNEDPTTFGGGTKLEIK [SEQ ID NO: 3]
a VL domain (28C5 VL) comprising, consisting of or consisting essentially of; and the sequence:
LQQSGPGLVKPSQSLSLTCTVTGYSITSDSAWNWIRQFPGNRLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLRFAYWGQGTLVTVSA [SEQ ID NO: 4]
a VH domain comprising, consisting of or consisting essentially of (28C5 VH)
and (3) the sequence:
QTPSSLSASLGDRVTISCRASQDIKNYLNWYQQPGGTVKVLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQRGDTLPWTFGGGTKLEIK [SEQ ID NO: 5]
a VL domain (18E12 VL) comprising, consisting of or consisting essentially of; and the sequence:
LESGPGLVAPSQSLSITCTVSGFSLTNYDISWIRQPPGKGLEWLGVIWTSGGTNYNSAFMSRLSITKDNSESQVFLKMNGLQTDDTGIYYCVRGDGNFYLYNFDYWGQGTTLTVSS [SEQ ID NO: 6]
a VH domain comprising, consisting of or consisting essentially of (18E12 VH)
The pharmaceutical composition according to any one of claims 32-41, selected from antibodies comprising 43. The pharmaceutical composition of claim 42, wherein said CD14 antagonist antibody comprises, consists of or consists essentially of the VL and VH CDR sequences of said antibody as defined in claim 10. The CD14 antagonist antibody is
(1) a) comprising L-CDR1, L-CDR2 and L-CDR3, where L-CDR1 comprises the sequence RASESVDSFGNSFMH [SEQ ID NO: 7] (3C10 L-CDR1); L-CDR2 comprises the sequence RAANLES [SEQ ID NO: 8] (3C10 L-CDR2); and L-CDR3 comprises the sequence QQSYEDPWT [SEQ ID NO:9] (3C10 L-CDR3); and b) H-CDR1, H- comprising CDR2 and H-CDR3, with H-CDR1 comprising the sequence SYAMS [SEQ ID NO: 10] (3C10 H-CDR1); H-CDR2 comprising the sequence SISSGGTTYYPDNVKG [SEQ ID NO: 11] (3C10 H-CDR2); and an antibody comprising an antibody VH domain, or an antigen-binding fragment thereof, wherein H-CDR3 comprises the sequence GYYDYHY [SEQ ID NO: 12] (3C10 H-CDR3);
(2) a) comprising L-CDR1, L-CDR2 and L-CDR3, where L-CDR1 comprises the sequence RASESVDSYVNSFLH [SEQ ID NO:13] (28C5 L-CDR1); L-CDR2 comprises the sequence RASNLQS [SEQ ID NO:14] (28C5 L-CDR2); and L-CDR3 comprises the sequence QQSNEDPTT [SEQ ID NO: 15] (28C5 L-CDR3); and b) H-CDR1, H- comprising CDR2 and H-CDR3, with H-CDR1 comprising the sequence SDSAWN [SEQ ID NO: 16] (28C5 H-CDR1); H-CDR2 comprising the sequence YISYSGSTSYNPSLKS [SEQ ID NO: 17] (28C5 H-CDR2); and an antibody comprising an antibody VH domain, or an antigen-binding fragment thereof, wherein H-CDR3 comprises the sequence GLRFAY [SEQ ID NO: 18] (28C5 H-CDR3); and (3) a) L-CDR1, L-CDR2 and L- L-CDR1 comprises the sequence RASQDIKNYLN [SEQ ID NO:19] (18E12 L-CDR1); L-CDR2 comprises the sequence YTSRLHS [SEQ ID NO:20] (18E12 L-CDR2); an antibody VL domain, or antigen-binding fragment thereof, comprising the sequence QRGDTLPWT [SEQ ID NO: 21] (18E12 L-CDR3); and b) comprising H-CDR1, H-CDR2 and H-CDR3, wherein H-CDR1 comprises the sequence NYDIS [ SEQ ID NO:22] (18E12 H-CDR1); H-CDR2 contains the sequence VIWTSGGTNYNSAFMS [SEQ ID NO:23] (18E12 H-CDR2); and H-CDR3 contains the sequence GDGNFYLYNFDY [SEQ ID NO:24] (18E12 H- 44. The pharmaceutical composition according to claim 43, selected from antibodies comprising an antibody VH domain, or an antigen-binding fragment thereof, comprising CDR3). 45. The pharmaceutical composition of claim 44, wherein said CD14 antagonist antibody is humanized. said CD14 antagonist antibody comprises a VL domain and a VH domain,
The VL domain has the amino acid sequence:
METDTILLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRASNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 25]
and said VH domain has the amino acid sequence:
MKVLSLLYLLTAIPGILSDVQLQSGPGLVKPSQSLSLTCTVTGYSITSDSSAWNWIRQFPGNRLEWMGYISYSGSTSSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLRFAYWGQGTLVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK [SEQ ID NO: 26]
including,
46. A pharmaceutical composition according to claim 45. 47. The pharmaceutical composition of Claim 46, wherein said CD14 antagonist antibody is IC14, or an antigen-binding fragment thereof. 47. The pharmaceutical composition of any one of claims 31-46, wherein said pharmaceutical composition further comprises an adjunctive anti-neurodegenerative agent, said adjunct treating said disease or symptom thereof. 49. The pharmaceutical composition of claim 48, wherein said adjunctive anti-neurodegenerative agent is an anti-inflammatory agent. 50. The pharmaceutical composition of Claim 49, wherein said anti-inflammatory agent is a complement inhibitor. 51. The pharmaceutical composition of Claim 50, wherein said complement inhibitor is a C5a inhibitor. 52. The pharmaceutical composition of claim 51, wherein said C5a inhibitor is PMX205 or eculizumab. 49. The pharmaceutical composition of claim 48, wherein said adjunctive anti-neurodegenerative agent is an agent that blocks the interaction of CD40 with CD40 ligand. 54. The pharmaceutical composition of claim 53, wherein said agent is an antibody that specifically binds CD40 and/or CD40 ligand. 55. The pharmaceutical composition of claim 54, wherein said antibody is AT-1502. 49. The pharmaceutical composition of claim 48, wherein said adjunctive anti-neurodegenerative agent is riluzole or edaravone.

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